Inir12 transgenic maize

ABSTRACT

Transgenic INIR12 maize plants comprising a vip3Aa19 or vip3Aa20 expression cassette linked to a secondary nopaline synthase terminator element which lack a selectable marker gene and/or which comprise modifications that provide for facile excision of the INIR12 transgenic locus from the maize plant genome are provided. Genomic DNA of INIR12 transgenic plants, detection of INIR12 plants and products thereof, methods of making INIR12 plants, and use of INIR12 plants to facilitate breeding are disclosed.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing contained in the file named “10085US3_ST25.txt”,which is 152,389 bytes as measured in the Windows operating system, andwhich was created on May 20, 2021 and electronically filed via EFS-Webon May 20, 2021, is incorporated herein by reference in its entirety.

BACKGROUND

Transgenes which are placed into different positions in the plant genomethrough non-site specific integration can exhibit different levels ofexpression (Weising et al., 1988, Ann. Rev. Genet. 22:421-477). Suchtransgene insertion sites can also contain various undesirablerearrangements of the foreign DNA elements that include deletions and/orduplications. Furthermore, many transgene insertion sites can alsocomprise selectable or scoreable marker genes which in some instancesare no longer required once a transgenic plant event containing thelinked transgenes which confer desirable traits are selected.

Commercial transgenic plants typically comprise one or more independentinsertions of transgenes at specific locations in the host plant genomethat have been selected for features that include expression of thetransgene(s) of interest and the transgene-conferred trait(s), absenceor minimization of rearrangements, and normal Mendelian transmission ofthe trait(s) to progeny. An example of a selected transgenic maize eventwhich confers tolerance to certain lepidopteran insect pests is theMIR162 transgenic maize event disclosed in U.S. Pat. No. 8,455,720.MIR162 transgenic maize plants express a VIP3Aa20 protein which canconfer resistance to fall armyworm (Spodoptera frugiperda), corn earworm(Helicoverpa zea), western bean cutworm (Striacosta albicosta), andblack cutworm (Agrotis ipsilon) infestations. MIR162 transgenic maizeplants also express a phosphomannose isomerase selectable markerprotein.

Methods for removing selectable marker genes and/or duplicatedtransgenes in transgene insertion sites in plant genomes involving useof site-specific recombinase systems (e.g., cre-lox) as well as forinsertion of new genes into transgene insertion sites have beendisclosed (Srivastava and Ow; Methods Mol Biol, 2015, 1287:95-103; Daleand Ow, 1991, Proc. Natl Acad. Sci. USA 88, 10558-10562; Srivastava andThomson, Plant Biotechnol J, 2016; 14(2):471-82). Such methods typicallyrequire incorporation of the recombination site sequences recognized bythe recombinase at particular locations within the transgene.

SUMMARY

Transgenic maize plant cells comprising a first ZmUbiInt promoter, avip3Aa19 or vip3Aa20 coding region which is operably linked to saidpromoter, a CaMV 35S terminator element which is operably linked to saidvip3Aa19 or vip3Aa20 coding region, and a nopaline synthase terminatorelement, wherein said cell does not contain a second ZmUbiInt promoterand an operably linked phosphomannose isomerase coding region betweensaid terminator elements are provided. Transgenic maize plant cellcomprising a nucleotide sequence comprising a first ZmUbiInt promoter, avip3Aa19 or vip3Aa20 coding region which is operably linked to saidpromoter, a CaMV 35S terminator element which is operably linked to saidvip3Aa19 or vip3Aa20 coding region, and a nopaline synthase terminatorelement, wherein said nucleotide sequence does not contain a secondZmUbiInt promoter and an operably linked phosphomannose isomerase codingregion between said terminator elements are provided. Transgenic maizeplant cells comprising a nucleotide sequence comprising a ZmUbiIntpromoter, a vip3Aa19 or vip3Aa20 coding region which is operably linkedto said promoter, a CaMV 35S terminator element which is operably linkedto said vip3Aa19 or vip3Aa20 coding region, and a nopaline synthaseterminator element, wherein said nucleotide sequence does not contain aphosphomannose isomerase coding region between said terminator elementsare provided. Transgenic maize plant cells comprising a nucleotidesequence comprising a first ZmUbiInt promoter, a vip3Aa19 or vip3Aa20coding region which is operably linked to said promoter, a CaMV 35Sterminator element which is operably linked to said vip3Aa19 or vip3Aa20coding region, and a nopaline synthase terminator element, wherein saidnucleotide sequence does not contain a second ZmUbiInt promoter and anoperably linked phosphomannose isomerase coding region are provided.Transgenic maize plant cell comprising a nucleotide sequence comprisinga ZmUbiInt promoter, a vip3Aa19 or vip3Aa20 coding region which isoperably linked to said promoter, a CaMV 35S terminator element which isoperably linked to said vip3Aa19 or vip3Aa20 coding region, and anopaline synthase terminator element, wherein said nucleotide sequencedoes not contain a phosphomannose isomerase coding region are provided.In certain embodiments, aforementioned transgenic maize plant cellswherein: (i) the ZmUbiInt promoter, the vip3Aa19 or vip3Aa20 codingregion which is operably linked to said promoter, the CaMV 35Sterminator element which is operably linked to said vip3Aa19 or vip3Aa20coding region are located in the maize plant cell genomic location ofthe MIR162 transgenic locus; (ii) wherein a selectable marker orscoreable is absent from said maize plant cell genomic location, and/or(iii) wherein the nopaline synthase terminator element is not separatedfrom the CaMV 35S terminator element by DNA encoding a selectable markerprotein, a scoreable marker protein, or a protein conferring a usefultrait are provided. Transgenic maize plant cells comprising an INIR12transgenic locus comprising the first ZmUbiInt promoter, the vip3Aa19 orvip3Aa20 coding region which is operably linked to said promoter, theCaMV 35S terminator element which is operably linked to said vip3Aa19 orvip3Aa20 coding region, and the nopaline synthase terminator element ofan original MIR162 transgenic locus allelic variants thereof, or othervariants thereof, wherein DNA of said original MIR162 transgenic locus,allelic variants thereof, or other variants thereof comprising a secondZmUbiInt promoter and an operably linked phosphomannose isomerase codingregion is absent are provided. In certain embodiments, the originalMIR162 transgenic locus is set forth in SEQ ID NO: 1, is present in seeddeposited at the ATCC under accession No. PTA-8166 (SEQ ID NO: 46) orprogeny thereof, is an allelic variant thereof, or is another variantthereof. In certain embodiments, an INIR12 transgenic locus comprises orfurther comprises an insertion and/or substitution of a DNA elementcomprising a cognate guide RNA recognition site (CgRRS) in a junctionpolynucleotide of said INIR12 transgenic locus, wherein the CgRRSoptionally comprises SEQ ID NO: 37. In certain embodiments, transgenicmaize plant cells comprising a INIR12 transgenic locus set forth in SEQID NO: 2, 3, 4, 5, 6, 29, 43, 44, 45, or 47 are provided. Also providedare transgenic maize plants and parts thereof including seeds whichcomprise the aforementioned transgenic maize plant cells. INIR12transgenic maize plants provided herein can exhibit resistance to fallarmyworm (Spodoptera frugiperda), corn earworm (Helicoverpa zea),western bean cutworm (Striacosta albicosta), and black cutworm (Agrotisipsilon) infestations in comparison to control maize plants which lackthe Vip3Aa protein.

Methods or obtaining a bulked population of inbred seed comprisingselfing any of the aforementioned INIR12 transgenic maize plants andharvesting seed comprising the INIR12 transgenic locus from the selfedmaize plant are provided.

Methods of obtaining hybrid maize seed comprising crossing any of theaforementioned INIR12 transgenic maize plant to a second maize plantwhich is genetically distinct from the first maize plant and harvestingseed comprising the INIR12 transgenic locus from the cross are provided.

DNA molecules comprising any one of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 25, 37, 40, 41, 42, 43, 44, 45, or 47 areprovided. Processed transgenic maize plant products and biologicalsamples comprising the aforementioned DNA molecules are also provided.Methods of detecting a maize plant cell comprising a INIR12 transgeniclocus comprising the step of detecting a DNA molecule comprising SEQ IDNO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 25, 37, 39, 40, 41,42, 43, 44, 45, or 47 are also provided.

Also provided are methods of excising a INIR12 transgenic locuscomprising an CgRRS and an originator guide RNA recognition site (OgRRS)from the genome of a maize plant cell comprising the steps of: (a)contacting a transgenic plant genome of a maize plant cell comprisingthe INIR12 transgenic locus with: (i) an RNA dependent DNA endonuclease(RdDe); and (ii) a guide RNA (gRNA) capable of hybridizing to the guideRNA hybridization site of the OgRRS and the CgRRS; wherein the RdDerecognizes a OgRRS/gRNA and a CgRRS/gRNA hybridization complex; and, (b)selecting a transgenic plant cell, transgenic plant part, or transgenicplant wherein the INIR12 transgenic locus flanked by the OgRRS and theCgRRS has been excised.

Also provided are methods of making transgenic maize plant cellcomprising an INIR12 transgenic locus comprising: (a) contacting thetransgenic plant genome of a maize MIR162 plant cell with: (i) a firstset of gene editing molecules comprising a first site-specific nucleasewhich introduces a first double stranded DNA break in a 5′ junctionpolynucleotide of an MIR162 transgenic locus; and (ii) a second set ofgene editing molecules comprising a second site-specific nuclease whichintroduces a second double stranded DNA break between the CaMV35Sterminator element and the ZmUbi promoter of said MIR162 transgeniclocus which is operably linked to DNA encoding a phosphomannoseisomerase (pmi) and a third site specific nuclease which introduces athird double stranded DNA break between the DNA encoding the pmi and DNAencoding the nopaline synthase (nos) terminator element of said MIR162transgenic locus; and (b) selecting a transgenic maize plant cell,transgenic maize callus, and/or a transgenic maize plant comprising anINIR12 transgenic locus wherein one or more nucleotides of said 5′junction polynucleotide have been deleted and/or substituted, whereinthe first ZmUbiInt promoter, the vip3Aa19 or vip3Aa20 coding regionwhich is operably linked to the first ZmUbiInt promoter, the CaMV 35Sterminator element which is operably linked to said vip3Aa19 or vip3Aa20coding region, and the nos terminator element of said MIR162 transgeniclocus are present, and wherein DNA of said MIR162 transgenic locuscomprising a second ZmUbiInt promoter and an operably linkedphosphomannose isomerase coding region is absent, thereby making atransgenic maize plant cell comprising an INIR12 transgenic locus.Transgenic maize plant cells, transgenic maize plant callus, transgenicmaize plants, and transgenic maize plant seeds comprising an INIR12transgenic locus made by the aforementioned methods are also provided.Also provided are methods of modifying a transgenic maize plant cellcomprising: obtaining a MIR162 maize event plant cell, a representativesample of which was deposited at the ATCC under accession No. PTA-8166,comprising a nucleotide sequence comprising a first ZmUbiInt promoter, avip3Aa19 or vip3Aa20 coding region which is operably linked to saidpromoter, a CaMV 35S terminator element which is operably linked to saidvip3Aa19 or vip3Aa20 coding region, a second ZmUbiInt promoter and anoperably linked phosphomannose isomerase coding region, and a nopalinesynthase terminator element; and modifying said nucleotide sequence toeliminate functionality of said phosphomannose isomerase coding regionand/or to substantially, essentially, or completely remove saidphosphomannose isomerase coding region, and optionally to eliminatefunctionality of, or substantially, essentially, or completely remove,said second ZmUbiInt promoter. Also provided are methods of modifying atransgenic maize plant cell comprising: obtaining a MIR162 maize eventplant cell, a representative sample of which was deposited at the ATCCunder accession No. PTA-8166, comprising a nucleotide sequencecomprising a first ZmUbiInt promoter, a vip3Aa19 or vip3Aa20 codingregion which is operably linked to said promoter, a CaMV 35S terminatorelement which is operably linked to said vip3Aa19 or vip3Aa20 codingregion, a second ZmUbiInt promoter and an operably linked phosphomannoseisomerase coding region, and a nopaline synthase terminator element; andmodifying said nucleotide sequence to substantially, essentially, orcompletely remove said phosphomannose isomerase coding region, andoptionally substantially, essentially, or completely remove said secondZmUniInt promoter.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 shows a diagram of transgene expression cassettes and selectablemarkers in the MIR162 transgenic locus in the deposited seed of ATCCaccession No. PTA-8166.

FIG. 2 shows a schematic diagram which compares current breedingstrategies for introgression of transgenic events (i.e., transgenicloci) to alternative breeding strategies for introgression of transgenicevents where the transgenic events (i.e., transgenic loci) can beremoved following introgression to provide different combinations oftransgenic traits. In FIG. 2, “GE” refers to genome editing (e.g.,including introduction of targeted genetic changes with genome editingmolecules and “Event Removal” refers to excision of a transgenic locus(i.e., an “Event”) or portion thereof with genome editing molecules.

FIG. 3A, B, C. FIG. 3A shows a schematic diagram of a non-limitingexample of: (i) an untransformed plant chromosome containingnon-transgenic DNA which includes the originator guide RNA recognitionsite (OgRRS) (top); (ii) the original transgenic locus with the OgRRS inthe non-transgenic DNA of the 1^(st) junction polynucleotide (middle);and (iii) the modified transgenic locus with a cognate guide RNAinserted into the non-transgenic DNA of the 2^(nd) junctionpolynucleotide (bottom). FIG. 3B shows a schematic diagram of anon-limiting example of a process where a modified transgenic locus witha cognate guide RNA inserted into the non-transgenic DNA of the 2^(nd)junction polynucleotide (top) is subjected to cleavage at the OgRRS andCgRRS with one guide RNA (gRNA) that hybridizes to gRNA hybridizationsite in both the OgRRS and the CgRRS and an RNA dependent DNAendonuclease (RdDe) that recognizes and cleaves the gRNA/OgRRS and thegRNA/CgRRS complex followed by non-homologous end joining processes toprovide a plant chromosome where the transgenic locus is excised. FIG.3C shows a schematic diagram of a non-limiting example of a processwhere a modified transgenic locus with a cognate guide RNA inserted intothe non-transgenic DNA of the 2^(nd) junction polynucleotide (top) issubjected to cleavage at the OgRRS and CgRRS with one guide RNA (gRNA)that hybridizes to the gRNA hybridization site in both the OgRRS and theCgRRS and an RNA dependent DNA endonuclease (RdDe) that recognizes andcleaves the gRNA/OgRRS and the gRNA/CgRRS complex in the presence of adonor DNA template. In FIG. 3C, cleavage of the modified transgeniclocus in the presence of the donor DNA template which has homology tonon-transgenic DNA but lacks the OgRRS in the 1^(st) and 2^(nd) junctionpolynucleotides followed by homology-directed repair processes toprovide a plant chromosome where the transgenic locus is excised andnon-transgenic DNA present in the untransformed plant chromosome is atleast partially restored.

FIG. 4 shows a schematic diagram of the hybridization sites for gRNAs ofSEQ ID NO: 20, 21, and 22. The 5′ junction polynucleotide sequence setforth in FIG. 4 corresponds to nucleotides 920 to 1240 of SEQ ID NO: 1and SEQ ID NO: 46.

FIGS. 5A, 5B, and 5C show the sequence (SEQ ID NO: 46) of the MIR162locus in the deposited seed of ATCC accession No. PTA-8166 which encodesthe Vip3Aa20 protein. FIG. 5A shows the 5′ end of the SEQ ID NO: 46sequence, FIG. 5B shows the internal SEQ ID NO: 46 sequence, and FIG. 5Cshows the 3′ end of the SEQ ID NO: 46 sequence. The endogenous genomicDNA (uppercase), transgenic insert DNA (lowercase) and 5′ and 3′junction sequences at both ends of the transgenic insert DNA are shown.The OgRRS sequence in the 3′ junction sequence (comprising SEQ ID NO: 27in the complementary DNA strand) is shown in bold and underlined.

FIGS. 6A, 6B, and 6C show the sequence (SEQ ID NO: 47) of an INIR12transgenic locus obtained by insertion of a CgRRS in the 5′ DNA junctionpolynucleotide sequence of an MIR 162 transgenic locus in the ATCCaccession no. PTA-8166 deposit which encodes the Vip3Aa20 protein. FIG.6A shows the 5′ end of the SEQ ID NO: 47 sequence, FIG. 6B shows theinternal SEQ ID NO: 47 sequence, and FIG. 6C shows the 3′ end of the SEQID NO: 47 sequence. The endogenous genomic DNA (uppercase) andtransgenic insert DNA (lowercase) as well as the 5′ and 3′ junctionsequences at both ends of the inserted transgenic DNA are shown. TheCgRRS sequence comprising the PAM site and gRNA hybridization site inthe 5′ junction polynucleotide sequence is shown in bold, lowercase, andunderlined. The OgRRS sequence in the 3′ junction polynucleotidesequence (comprising SEQ ID NO: 27 in the complementary DNA strand) isshown in bold, uppercase, and underlined.

DETAILED DESCRIPTION

Unless otherwise stated, nucleic acid sequences in the text of thisspecification are given, when read from left to right, in the 5′ to 3′direction. Nucleic acid sequences may be provided as DNA or as RNA, asspecified; disclosure of one necessarily defines the other, as well asnecessarily defines the exact complements, as is known to one ofordinary skill in the art.

Where a term is provided in the singular, the inventors also contemplateembodiments described by the plural of that term.

The term “about” as used herein means a value or range of values whichwould be understood as an equivalent of a stated value and can begreater or lesser than the value or range of values stated by 10percent. Each value or range of values preceded by the term “about” isalso intended to encompass the embodiment of the stated absolute valueor range of values.

The phrase “allelic variant” as used herein refers to a polynucleotideor polypeptide sequence variant that occurs in a different strain,variety, or isolate of a given organism.

The term “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term and/or” as used in a phrase such as “Aand/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C;A and C; A and B; B and C; A (alone); B (alone); and C (alone).

As used herein, the phrase “approved transgenic locus” is a geneticallymodified plant event which has been authorized, approved, and/orde-regulated for any one of field testing, cultivation, humanconsumption, animal consumption, and/or import by a governmental body.Illustrative and non-limiting examples of governmental bodies whichprovide such approvals include the Ministry of Agriculture of Argentina,Food Standards Australia New Zealand, National Biosafety TechnicalCommittee (CTNBio) of Brazil, Canadian Food Inspection Agency, ChinaMinistry of Agriculture Biosafety Network, European Food SafetyAuthority, US Department of Agriculture, US Department of EnvironmentalProtection, and US Food and Drug Administration.

The term “backcross”, as used herein, refers to crossing an F1 plant orplants with one of the original parents. A backcross is used to maintainor establish the identity of one parent (species) and to incorporate aparticular trait from a second parent (species). The term “backcrossgeneration”, as used herein, refers to the offspring of a backcross.

As used herein, the phrase “biological sample” refers to either intactor non-intact (e.g. milled seed or plant tissue, chopped plant tissue,lyophilized tissue) plant tissue. It may also be an extract comprisingintact or non-intact seed or plant tissue. The biological sample cancomprise flour, meal, syrup, oil, starch, and cereals manufactured inwhole or in part to contain crop plant by-products. In certainembodiments, the biological sample is “non-regenerable” (i.e., incapableof being regenerated into a plant or plant part). In certainembodiments, the biological sample refers to a homogenate, an extract,or any fraction thereof containing genomic DNA of the organism fromwhich the biological sample was obtained, wherein the biological sampledoes not comprise living cells.

As used herein, the terms “correspond,” “corresponding,” and the like,when used in the context of an nucleotide position, mutation, and/orsubstitution in any given polynucleotide (e.g., an allelic variant ofSEQ ID NO: 1 or SEQ ID NO: 46) with respect to the referencepolynucleotide sequence (e.g., SEQ ID NO: 1 or SEQ ID NO: 46) all referto the position of the polynucleotide residue in the given sequence thathas identity to the residue in the reference nucleotide sequence whenthe given polynucleotide is aligned to the reference polynucleotidesequence using a pairwise alignment algorithm (e.g., CLUSTAL O 1.2.4with default parameters).

As used herein, the terms “Cpf1” and “Cas12a” are used interchangeablyto refer to the same RNA dependent DNA endonuclease (RdDe). Cas12aproteins include the protein provided herein as SEQ ID NO: 38.

The term “crossing” as used herein refers to the fertilization of femaleplants (or gametes) by male plants (or gametes). The term “gamete”refers to the haploid reproductive cell (egg or pollen) produced inplants by meiosis from a gametophyte and involved in sexualreproduction, during which two gametes of opposite sex fuse to form adiploid zygote. The term generally includes reference to a pollen(including the sperm cell) and an ovule (including the ovum). Whenreferring to crossing in the context of achieving the introgression of agenomic region or segment, the skilled person will understand that inorder to achieve the introgression of only a part of a chromosome of oneplant into the chromosome of another plant, random portions of thegenomes of both parental lines recombine during the cross due to theoccurrence of crossing-over events in the production of the gametes inthe parent lines. Therefore, the genomes of both parents must becombined in a single cell by a cross, where after the production ofgametes from the cell and their fusion in fertilization will result inan introgression event.

As used herein, the phrases “DNA junction polynucleotide” and “junctionpolynucleotide” refers to a polynucleotide of about 18 to about 500 basepairs in length comprised of both endogenous chromosomal DNA of theplant genome and heterologous transgenic DNA which is inserted in theplant genome. A junction polynucleotide can thus comprise about 8, 10,20, 50, 100, 200, 250, 500, or 1000 base pairs of endogenous chromosomalDNA of the plant genome and about 8, 10, 20, 50, 100, 200, 250, 500, or1000 base pairs of heterologous transgenic DNA which span the one end ofthe transgene insertion site in the plant chromosomal DNA. Transgeneinsertion sites in chromosomes will typically contain both a 5′ junctionpolynucleotide and a 3′ junction polynucleotide. In embodiments setforth herein in SEQ ID NO: 1, the 5′ junction polynucleotide is locatedat the 5′ end of the sequence and the 3′ junction polynucleotide islocated at the 3′ end of the sequence. In a non-limiting andillustrative example, a 5′ junction polynucleotide of a transgenic locusis telomere proximal in a chromosome arm and the 3′ junctionpolynucleotide of the transgenic locus is centromere proximal in thesame chromosome arm. In another non-limiting and illustrative example, a5′ junction polynucleotide of a transgenic locus is centromere proximalin a chromosome arm and the 3′ junction polynucleotide of the transgeniclocus is telomere proximal in the same chromosome arm. The junctionpolynucleotide which is telomere proximal and the junctionpolynucleotide which is centromere proximal can be determined bycomparing non-transgenic genomic sequence of a sequenced non-transgenicplant genome to the non-transgenic DNA in the junction polynucleotides.

The term “donor,” as used herein in the context of a plant, refers tothe plant or plant line from which the trait, transgenic event, orgenomic segment originates, wherein the donor can have the trait,introgression, or genomic segment in either a heterozygous or homozygousstate.

As used herein, the terms “excise” and “delete,” when used in thecontext of a DNA molecule, are used interchangeably to refer to theremoval of a given DNA segment or element (e.g., transgene element ortransgenic locus or portion thereof) of the DNA molecule.

As used herein, the phrase “elite crop plant” refers to a plant whichhas undergone breeding to provide one or more trait improvements. Elitecrop plant lines include plants which are an essentially homozygous,e.g. inbred or doubled haploid. Elite crop plants can include inbredlines used as is or used as pollen donors or pollen recipients in hybridseed production (e.g. used to produce F1 plants). Elite crop plants caninclude inbred lines which are selfed to produce non-hybrid cultivars orvarieties or to produce (e.g., bulk up) pollen donor or recipient linesfor hybrid seed production. Elite crop plants can include hybrid F1progeny of a cross between two distinct elite inbred or doubled haploidplant lines.

As used herein, an “event,” “a transgenic event,” “a transgenic locus”and related phrases refer to an insertion of one or more transgenes at aunique site in the genome of a plant as well as to DNA fragments, plantcells, plants, and plant parts (e.g., seeds) comprising genomic DNAcontaining the transgene insertion. Such events typically comprise botha 5′ and a 3′ DNA junction polynucleotide and confer one or more usefultraits including herbicide tolerance, insect resistance, male sterility,and the like.

As used herein, the phrases “endogenous sequence,” “endogenous gene,”“endogenous DNA,” “endogenous polynucleotide,” and the like refer to thenative form of a polynucleotide, gene or polypeptide in its naturallocation in the organism or in the genome of an organism.

The terms “exogenous” and “heterologous” as are used synonymously hereinto refer to any polynucleotide (e.g. DNA molecule) that has beeninserted into a new location in the genome of a plant. Non-limitingexamples of an exogenous or heterologous DNA molecule include asynthetic DNA molecule, a non-naturally occurring DNA molecule, a DNAmolecule found in another species, a DNA molecule found in a differentlocation in the same species, and/or a DNA molecule found in the samestrain or isolate of a species, where the DNA molecule has been insertedinto a new location in the genome of a plant.

As used herein, the term “F1” refers to any offspring of a cross betweentwo genetically unlike individuals.

The term “gene,” as used herein, refers to a hereditary unit consistingof a sequence of DNA that occupies a specific location on a chromosomeand that contains the genetic instruction for a particularcharacteristics or trait in an organism. The term “gene” thus includes anucleic acid (for example, DNA or RNA) sequence that comprises codingsequences necessary for the production of an RNA, or a polypeptide orits precursor. A functional polypeptide can be encoded by a full lengthcoding sequence or by any portion of the coding sequence as long as thedesired activity or functional properties (e.g., enzymatic activity,pesticidal activity, ligand binding, and/or signal transduction) of theRNA or polypeptide are retained.

The term “identifying,” as used herein with respect to a plant, refersto a process of establishing the identity or distinguishing character ofa plant, including exhibiting a certain trait, containing one or moretransgenes, and/or containing one or more molecular markers.

As used herein, the term “INIR12” is used herein to refer eitherindividually or collectively to items that include any or all of theMIR162 transgenic maize loci which have been modified as disclosedherein, transgenic maize plants and parts thereof including seed thatcomprise the modified MIR162 transgenic loci, and DNA obtainedtherefrom.

The term “isolated” as used herein means having been removed from itsnatural environment.

As used herein, the terms “include,” “includes,” and “including” are tobe construed as at least having the features to which they refer whilenot excluding any additional unspecified features.

As used herein, the phrase “introduced transgene” is a transgene notpresent in the original transgenic locus in the genome of an initialtransgenic event or in the genome of a progeny line obtained from theinitial transgenic event. Examples of introduced transgenes includeexogenous transgenes which are inserted in a resident originaltransgenic locus.

As used herein, the terms “introgression”, “introgressed” and“introgressing” refer to both a natural and artificial process, and theresulting plants, whereby traits, genes or DNA sequences of one species,variety or cultivar are moved into the genome of another species,variety or cultivar, by crossing those species. The process mayoptionally be completed by backcrossing to the recurrent parent.Examples of introgression include entry or introduction of a gene, atransgene, a regulatory element, a marker, a trait, a trait locus, or achromosomal segment from the genome of one plant into the genome ofanother plant.

The phrase “marker-assisted selection”, as used herein, refers to thediagnostic process of identifying, optionally followed by selecting aplant from a group of plants using the presence of a molecular marker asthe diagnostic characteristic or selection criterion. The processusually involves detecting the presence of a certain nucleic acidsequence or polymorphism in the genome of a plant.

As used herein, the term “MIR162” is used to refer to items that includea transgenic maize locus, transgenic maize plants and parts thereofincluding seed set forth in U.S. Pat. No. 8,455,720, which isincorporated herein by reference in its entirety. Representative MIR162transgenic maize seed have been deposited at the American Type CultureCollection (ATCC, Manassas, Va., USA) as accession No. PTA-8166. MIR162transgenic loci include loci having the sequence of SEQ ID NO:1, thesequence of the MIR162 locus in the deposited seed of accession No.PTA-8166 (SEQ ID NO: 46) and any progeny thereof, as well as allelicvariants and other variants of SEQ ID NO:1 or SEQ ID NO: 46. Othervariants of a MIR162 locus can include variants in MIR162 other thanthose disclosed herein obtained by gene editing techniques (e.g., by useof RdDe, CBE, or ABE and gRNAs, TALENs, and/or ZFN).

The phrase “molecular marker”, as used herein, refers to an indicatorthat is used in methods for visualizing differences in characteristicsof nucleic acid sequences. Examples of such indicators are restrictionfragment length polymorphism (RFLP) markers, amplified fragment lengthpolymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs),microsatellite markers (e.g. SSRs), sequence-characterized amplifiedregion (SCAR) markers, Next Generation Sequencing (NGS) of a molecularmarker, cleaved amplified polymorphic sequence (CAPS) markers or isozymemarkers or combinations of the markers described herein which defines aspecific genetic and chromosomal location.

As used herein the terms “native” or “natural” define a condition foundin nature. A “native DNA sequence” is a DNA sequence present in naturethat was produced by natural means or traditional breeding techniquesbut not generated by genetic engineering (e.g., using molecularbiology/transformation techniques).

The term “offspring”, as used herein, refers to any progeny generationresulting from crossing, selfing, or other propagation technique.

The phrase “operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. For instance, a promoter is operablylinked to a coding sequence if the promoter affects its transcription orexpression. When the phrase “operably linked” is used in the context ofa PAM site and a guide RNA hybridization site, it refers to a PAM sitewhich permits cleavage of at least one strand of DNA in a polynucleotidewith an RNA dependent DNA endonuclease or RNA dependent DNA nickasewhich recognize the PAM site when a guide RNA complementary to guide RNAhybridization site sequences adjacent to the PAM site is present. AOgRRS and its CgRRS, sPAM sites, or sigRNAR sites are operably linked tojunction polynucleotides when they can be recognized by a gRNA and anRdDe to provide for excision of the transgenic locus or portion thereofflanked by the junction polynucleotides. When the phrase “operablylinked” is used in the context of a signature PAM site and a DNAjunction polynucleotide, it refers to a PAM site which permits cleavageof at least one strand of DNA in the junction polynucleotide with an RNAdependent DNA endonuclease, RNA dependent DNA binding protein, or RNAdependent DNA nickase which recognizes the PAM site when a guide RNAcomplementary to sequences adjacent to the PAM site is present. When thephrase “operably linked” is used in the context of a sigRNAR site and aDNA junction polynucleotide, it refers to a sigRNAR site which permitscleavage of at least one strand of DNA in the junction polynucleotidewith an RNA dependent DNA endonuclease, RNA dependent DNA bindingprotein, or RNA dependent DNA nickase which recognizes the sigRNAR sitewhen a guide RNA complementary to the heterologous sequences adjacent inthe sigRNAR site is present.

As used herein, the term “plant” includes a whole plant and anydescendant, cell, tissue, or part of a plant. The term “plant parts”include any part(s) of a plant, including, for example and withoutlimitation: seed (including mature seed and immature seed); a plantcutting; a plant cell; a plant cell culture; or a plant organ (e.g.,pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, andexplants). A plant tissue or plant organ may be a seed, protoplast,callus, or any other group of plant cells that is organized into astructural or functional unit. A plant cell or tissue culture may becapable of regenerating a plant having the physiological andmorphological characteristics of the plant from which the cell or tissuewas obtained, and of regenerating a plant having substantially the samegenotype as the plant. Regenerable cells in a plant cell or tissueculture may be embryos, protoplasts, meristematic cells, callus, pollen,leaves, anthers, roots, root tips, silk, flowers, kernels, ears, cobs,husks, or stalks. In contrast, some plant cells are not capable of beingregenerated to produce plants and are referred to herein as“non-regenerable” plant cells.

The term “purified,” as used herein defines an isolation of a moleculeor compound in a form that is substantially free of contaminantsnormally associated with the molecule or compound in a native or naturalenvironment and means having been increased in purity as a result ofbeing separated from other components of the original composition. Theterm “purified nucleic acid” is used herein to describe a nucleic acidsequence which has been separated from other compounds including, butnot limited to polypeptides, lipids and carbohydrates.

The term “recipient”, as used herein, refers to the plant or plant linereceiving the trait, transgenic event or genomic segment from a donor,and which recipient may or may not have the have trait, transgenic eventor genomic segment itself either in a heterozygous or homozygous state.

As used herein the term “recurrent parent” or “recurrent plant”describes an elite line that is the recipient plant line in a cross andwhich will be used as the parent line for successive backcrosses toproduce the final desired line.

As used herein the term “recurrent parent percentage” relates to thepercentage that a backcross progeny plant is identical to the recurrentparent plant used in the backcross. The percent identity to therecurrent parent can be determined experimentally by measuring geneticmarkers such as SNPs and/or RFLPs or can be calculated theoreticallybased on a mathematical formula.

The terms “selfed,” “selfing,” and “self,” as used herein, refer to anyprocess used to obtain progeny from the same plant or plant line as wellas to plants resulting from the process. As used herein, the terms thusinclude any fertilization process wherein both the ovule and pollen arefrom the same plant or plant line and plants resulting therefrom.Typically, the terms refer to self-pollination processes and progenyplants resulting from self-pollination.

The term “selecting”, as used herein, refers to a process of picking outa certain individual plant from a group of individuals, usually based ona certain identity, trait, characteristic, and/or molecular marker ofthat individual.

As used herein, the phrase “originator guide RNA recognition site” orthe acronym “OgRRS” refers to an endogenous DNA polynucleotidecomprising a protospacer adjacent motif (PAM) site operably linked to aguide RNA hybridization site. In certain embodiments, an OgRRS can belocated in an untransformed plant chromosome or in non-transgenic DNA ofa DNA junction polynucleotide of both an original transgenic locus and amodified transgenic locus. In certain embodiments, an OgRRS can belocated in transgenic DNA of a DNA junction polynucleotide of both anoriginal transgenic locus and a modified transgenic locus. In certainembodiments, an OgRRS can be located in both transgenic DNA andnon-transgenic DNA of a DNA junction polynucleotide of both an originaltransgenic locus and a modified transgenic locus (i.e., can spantransgenic and non-transgenic DNA in a DNA junction polynucleotide).

As used herein the phrase “cognate guide RNA recognition site” or theacronym “CgRRS” refer to a DNA polynucleotide comprising a PAM siteoperably linked to a guide RNA hybridization site, where the CgRRS isabsent from transgenic plant genomes comprising a first originaltransgenic locus that is unmodified and where the CgRRS and itscorresponding OgRRS can hybridize to a single gRNA. A CgRRS can belocated in transgenic DNA of a DNA junction polynucleotide of a modifiedtransgenic locus, in transgenic DNA of a DNA junction polynucleotide ofa modified transgenic locus, or in both transgenic and non-transgenicDNA of a modified transgenic locus (i.e., can span transgenic andnon-transgenic DNA in a DNA junction polynucleotide).

As used herein, the phrase “a transgenic locus excision site” refers tothe DNA which remains in the genome of a plant or in a DNA molecule(e.g., an isolated or purified DNA molecule) wherein a segmentcomprising, consisting essentially of, or consisting of a transgeniclocus or portion thereof has been deleted. In a non-limiting andillustrative example, a transgenic locus excision site can thus comprisea contiguous segment of DNA comprising at least 10 base pairs of DNAthat is telomere proximal to the deleted transgenic locus or to thedeleted segment of the transgenic locus and at least 10 base pairs ofDNA that is centromere proximal to the deleted transgenic locus or tothe deleted segment of the transgenic locus.

As used herein, the phrase “signature protospacer adjacent motif (sPAM)”or acronym “sPAM” refer to a PAM which has been introduced into atransgenic plant genome by genome editing, wherein the sPAM is absentfrom a transgenic plant genome comprising the original transgenic locus.An sPAM can be introduced by an insertion, deletion, and or substitutionof one or more nucleotides in genomic DNA.

As used herein the phrase “signature guide RNA Recognition site” oracronym “sigRNAR site” refer to a DNA polynucleotide comprising aheterologous crRNA (CRISPR RNA) binding sequence located immediately 5′or 3′ to a PAM site, wherein the sigRNAR site has been introduced into atransgenic plant genome by genome editing and wherein at least theheterologous crRNA binding sequence is absent from a transgenic plantgenome comprising the original transgenic locus. In certain embodiments,the heterologous crRNA binding sequence is operably linked to apre-existing PAM site in the transgenic plant genome. In otherembodiments, the heterologous crRNA binding sequence is operably linkedto a sPAM site in the transgenic plant genome.

As used herein, the phrase “transgene element” refers to a segment ofDNA comprising, consisting essentially of, or consisting of a promoter,a 5′ UTR, an intron, a coding region, a 3′UTR, or a polyadenylationsignal. Polyadenylation signals include transgene elements referred toas “terminators” (e.g., NOS, pinII, rbcs, Hsp17, TubA).

To the extent to which any of the preceding definitions is inconsistentwith definitions provided in any patent or non-patent referenceincorporated herein by reference, any patent or non-patent referencecited herein, or in any patent or non-patent reference found elsewhere,it is understood that the preceding definition will be used herein.

Genome editing molecules can permit introduction of targeted geneticchange conferring desirable traits in a variety of crop plants (Zhang etal. Genome Biol. 2018; 19: 210; Schindele et al. FEBS Lett. 2018;592(12):1954). Desirable traits introduced into crop plants such asmaize include herbicide tolerance, improved food and/or feedcharacteristics, male-sterility, and drought stress tolerance.Nonetheless, full realization of the potential of genome editing methodsfor crop improvement will entail efficient incorporation of the targetedgenetic changes in germplasm of different elite crop plants adapted fordistinct growing conditions. Such elite crop plants will also desirablycomprise useful transgenic loci which confer various traits includingherbicide tolerance, pest resistance (e.g.; insect, nematode, fungaldisease, and bacterial disease resistance), conditional male sterilitysystems for hybrid seed production, abiotic stress tolerance (e.g.,drought tolerance), improved food and/or feed quality, and improvedindustrial use (e.g., biofuel).

INIR12 transgenic loci comprising modifications of a MIR162 transgenicloci in a maize plant genome by directed insertion, deletion, and/orsubstitution of DNA within or adjacent to such MIR162 transgenic loci aswell as methods of making and using such INIR12 transgenic loci areprovided herein. In certain embodiments, the INIR12 transgenic locicomprise the first ZmUbiInt promoter, the vip3Aa19 or vip3Aa20 codingregion which is operably linked to said promoter, the CaMV 35Sterminator element which is operably linked to said vip3Aa19 or vip3Aa20coding region, and the nopaline synthase terminator element of an MIR162transgenic locus, wherein DNA of said MIR162 transgenic locus comprisinga second ZmUbiInt promoter and an operably linked phosphomannoseisomerase (pmi) coding region is absent. Such INIR12 transgenic loci canthus comprise a vip3Aa19 or vip3Aa20 expression cassette having twotandemly arrayed terminator elements (i.e., a CaMV35S and a NOSterminator) while lacking non-essential DNA elements (i.e., theduplicate copy of the ZmUbiInt promoter and pmi selectable marker genewhich is operably linked thereto).

In certain embodiments, INIR12 transgenic loci provided herein can thuscomprise deletions of selectable marker genes and/or repetitivesequences. In its unmodified form (in certain embodiments, the“unmodified form” is the “original form,” “original transgenic locus,”etc.) a MIR162 transgenic locus comprises a phosphomannose isomerase(pmi)-encoding selectable marker gene which confers the ability to growon mannose as a carbon source. In embodiments provided herein, theselectable marker gene which is deleted comprises, consists essentiallyof, or consists of a DNA molecule encoding: (i) the phosphomannoseisomerase (pmi) of a MIR162 transgenic locus and the ZmUbi promoter thatis operably linked thereto; or (ii) the phosphomannose isomerase (pmi)of a MIR162 transgenic locus and both the ZmUbi promoter and NOSterminator that are operably linked thereto. In certain embodiments, DNAelements comprising the ZmUbi promoter and operably linked pmi codingregion corresponding to at least nucleotides 5837, 5838, 5840, 5845, or5850 to 8040, 9060, 9080, 9090, 9100, or 9105 of SEQ ID NO:1 or SEQ IDNO:46 can be absent from an INIR12 locus. In certain embodiments, theINIR12 locus comprising a deletion of DNA encoding the pmi gene and theoperably linked ZmUbi promoter is set forth in SEQ ID NO: 2, whereinnucleotides designated n in the sequence are either absent,independently selected from a guanine, a cytosine, an adenine residue,or a thymine, comprise or consist of 1 or more nucleotides correspondingto nucleotides 5831 to 5836 of SEQ ID NO:1 or SEQ ID NO:46 and/orcomprise or consist of 1 or more nucleotides corresponding tonucleotides 9102 to 9107 of SEQ ID NO:1 or SEQ ID NO:46. In certainembodiments, the deletion junction sequence present in an INIR12transgenic locus comprises a DNA molecule set forth in SEQ ID NO: 25which corresponds to nucleotides 5821 to 5850 of SEQ ID NO: 6. Incertain embodiments, the DNA comprising the ZmUbi promoter and operablylinked pmi coding region to be deleted is flanked by operably linkedprotospacer adjacent motif (PAM) sites in a MIR162 transgenic locuswhich are recognized by an RNA dependent DNA endonuclease (RdDe); forexample, a class 2 type II or class 2 type V RdDe. In certainembodiments, the deleted selectable marker gene is replaced in an INIR12transgenic locus by an introduced DNA sequence as discussed in furtherdetail elsewhere herein. For example, in certain embodiments, theintroduced DNA sequence comprises a trait expression cassette such as atrait expression cassette of another transgenic locus. In addition tothe deletion of a selectable marker gene, in certain embodiments atleast one copy of a repetitive sequence has also been deleted withgenome editing molecules from an MIR162 transgenic locus. In certainembodiments, the repetitive sequence comprises, consists essentially of,or consists of the two distinct ZmUbiInt promoters which are eachoperably linked to the VIP3Aa20 gene and to the pmi selectable markergene within the MIR162 transgenic locus (e.g., as depicted in FIG. 1).In certain embodiments, the repetitive sequence which comprises,consists essentially of, or consists of the second ZmUbiInt promoter andwhich is operably linked to the pmi selectable maker of an MIR162transgenic locus is absent from the INIR12 transgenic locus. In certainembodiments, any of the aforementioned INIR12 transgenic loci canoptionally further comprise: (i) an OgRRS and a CgRRS which are operablylinked to a 1^(st) and a 2^(nd) junction sequence of the INIR12transgenic locus; (ii) one or more signature protospacer adjacent motif(sPAM) sites which are operably linked to a 1^(st) and a 2^(nd) junctionsequence of the INIR12 transgenic locus; or (iii) signature guide RNARecognition site (sigRNAR) sites which are operably linked to a 1^(st)and a 2^(nd) junction sequence of the INIR12 transgenic locus. Alsoprovided herein are plants comprising any of aforementioned INIR12transgenic loci.

In certain embodiments, an INIR12 transgenic locus can further comprisemodifications of a 5′ or 3′ junction polynucleotide of an MIR162transgenic locus (e.g., as set forth in SEQ ID NO:1 or SEQ ID NO:46 andin FIG. 1). Such modifications of junction polynucleotides includedeletions of DNA segments comprising non-essential transgenic DNA in thejunction polynucleotide. In certain embodiments, such deletions ofnon-essential DNA of a 5′ junction polynucleotide of an INIR12transgenic locus include those set forth in SEQ ID NO: 3, wherein one ormore nucleotides in a segment corresponding to nucleotides 1089 to 1098are absent or independently selected from A, C, G, or T, with theproviso that the nucleotides 1089 to 1098 of SEQ ID NO:3 are notidentical to nucleotides 1089 to 1098 of SEQ ID NO:1 or SEQ ID NO:46. Incertain embodiments, such deletions of non-essential DNA of a 5′junction polynucleotide of an INIR12 transgenic locus include thosewherein nucleotides corresponding to nucleotides 1081 to 1104 of SEQ IDNO:3 are: (i) each either absent or independently selected from aguanine, a cytosine, an adenine residue, or a thymine residue; (ii)comprise about 2 to 8 consecutive residues of nucleotides 1,081 to 1092of SEQ ID NO:1 or SEQ ID NO:46 and/or about 2 to 8 consecutive residuesof nucleotides 1093 to 1104 of SEQ ID NO:1 or SEQ ID NO:46; or (iii) anycombination of (i) and (ii), wherein each of (i), (ii), and (iii) arewith the proviso that the nucleotides corresponding to nucleotides 1081to 1104 of SEQ ID NO: 3 are not identical to nucleotides 1081 to 1104 ofSEQ ID NO:1 or SEQ ID NO:46. In certain embodiments, such deletions ofnon-essential DNA of a 5′ junction polynucleotide of an INIR12transgenic locus include those wherein nucleotides corresponding tonucleotides 1081 to 1104 of SEQ ID NO:3 are set forth in SEQ ID NO: 7,wherein n is absent, is independently selected from A, C, G, or T,correspond to 1 to 10 residues of nucleotides 1083 to 1092 of SEQ IDNO:1 or SEQ ID NO:46, and/or correspond to 1 to 10 residues ofnucleotides 1093 to 1102 of SEQ ID NO:1 or SEQ ID NO:46 with the provisothat nucleotides corresponding to nucleotide 3 to 22 of SEQ ID NO: 7 arenot identical to residues 1083 to 1102 of SEQ ID NO:1 or SEQ ID NO:46.In certain embodiments, such deletions of non-essential DNA of a 5′junction polynucleotide of an INIR12 transgenic locus include thosewherein nucleotides corresponding to nucleotides 1081 to 1104 of SEQ IDNO:3 are set forth in SEQ ID NO: 8, wherein n is absent, isindependently selected from A, C, G, or T, correspond to 1 to 5 residuesof nucleotides 1088 to 1092 of SEQ ID NO:1 or SEQ ID NO:46, and/orcorrespond to 1 to 5 residues of nucleotides 1093 to 1097 of SEQ ID NO:1or SEQ ID NO:46 with the proviso that nucleotides corresponding toresidues 8 to 17 of SEQ ID NO: 8 are not identical to residues 1088 to1097 of SEQ ID NO:1 or SEQ ID NO:46. In certain embodiments, suchdeletions of non-essential DNA of a 5′ junction polynucleotide of anINIR12 transgenic locus include those wherein nucleotides correspondingto nucleotides 1081 to 1104 of SEQ ID NO:3 are set forth in SEQ ID NO:9; wherein n is absent, is independently selected from A, C, G, or T,correspond to 1 to 3 residues of nucleotides 1090-1092 of SEQ ID NO:1 orSEQ ID NO:46, and/or correspond to 1 to 3 residues of nucleotides 1093to 1095 of SEQ ID NO:1 or SEQ ID NO:46 with the proviso that nucleotidescorresponding to residues 1090 to 1095 of SEQ ID NO: 9 are not identicalto nucleotides 1090 to 1095 of SEQ ID NO:1 or SEQ ID NO:46. In certainembodiments, such deletions of non-essential DNA of a 5′ junctionpolynucleotide of an INIR12 transgenic locus include those whereinnucleotides corresponding to nucleotides 1081 to 1104 of SEQ ID NO:3 areset forth in SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. Incertain embodiments, such deletions of non-essential DNA of a 5′junction polynucleotide of an INIR12 transgenic locus include those setforth in SEQ ID NO: 39 and 40.

Also provided herein are methods whereby targeted genetic changes areefficiently combined with desired subsets of transgenic loci in eliteprogeny plant lines (e.g., elite inbreds used for hybrid seed productionor for inbred varietal production). Examples of such methods includethose illustrated in FIG. 2. In certain embodiments, INIR12 transgenicloci provided here are characterized by polynucleotide sequences thatcan facilitate as necessary the removal of the INIR12 transgenic locifrom the genome. Useful applications of such INIR12 transgenic loci andrelated methods of making include targeted excision of a INIR12transgenic locus or portion thereof in certain breeding lines tofacilitate recovery of germplasm with subsets of transgenic traitstailored for specific geographic locations and/or grower preferences.Other useful applications of such INIR12 transgenic loci and relatedmethods of making include removal of transgenic traits from certainbreeding lines when it is desirable to replace the trait in the breedingline without disrupting other transgenic loci and/or non-transgenicloci. In certain embodiments, maize genomes containing INIR12 transgenicloci or portions thereof which can be selectively excised with one ormore gRNA molecules and RdDe (RNA dependent DNA endonucleases) whichform gRNA/target DNA complexes. Such selectively excisable INIR12transgenic loci can comprise an originator guide RNA recognition site(OgRRS) which is identified in non-transgenic DNA, transgenic DNA, or acombination thereof in of a first junction polynucleotide of thetransgenic locus and cognate guide RNA recognition site (CgRRS) which isintroduced (e.g., by genome editing methods) into a second junctionpolynucleotide of the transgenic locus and which can hybridize to thesame gRNA as the OgRRS, thereby permitting excision of the modifiedtransgenic locus or portions thereof with a single guide RNA (e.g., asshown in FIGS. 3A and B). In certain embodiments, an originator guideRNA recognition site (OgRRS) comprises endogenous DNA found inuntransformed plants and in endogenous non-transgenic DNA of junctionpolynucleotides of transgenic plants containing a modified or unmodifiedtransgenic locus. In certain embodiments, an originator guide RNArecognition site (OgRRS) comprises exogenous transgenic DNA of junctionpolynucleotides of transgenic plants containing a modified or unmodifiedtransgenic locus. The OgRRS located in non-transgenic DNA, transgenicDNA, or a combination thereof in of a first DNA junction polynucleotideis used to design a related cognate guide RNA recognition site (CgRRS)which is introduced (e.g., by genome editing methods) into the secondjunction polynucleotide of the transgenic locus. A CgRRS is thus presentin junction polynucleotides of modified transgenic loci provided hereinand is absent from endogenous DNA found in untransformed plants andabsent from junction sequences of transgenic plants containing anunmodified transgenic locus. A CgRRS is also absent from a combinationof non-transgenic and transgenic DNA found in junction sequences oftransgenic plants containing an unmodified transgenic locus. In certainembodiments such as those illustrated in the non-limiting example ofFIG. 3, the OgRRS is located in non-transgenic DNA of a 5′ junctionpolynucleotide and the CgRRS is introduced into non-transgenic DNA of a3′ junction polynucleotide. In other embodiments, the OgRRS can belocated in non-transgenic DNA of a 3′ junction polynucleotide and theCgRRS is introduced into non-transgenic DNA, transgenic DNA, or acombination thereof in a 5′ junction polynucleotide. Examples of OgRRSpolynucleotide sequences in or near a 3′ junction polynucleotide in anMIR162 transgenic locus include SEQ ID NO: 26, 27, and 28. OgRRSpolynucleotide sequences located in a first junction polynucleotide canbe introduced into the second junction polynucleotide using donor DNAtemplates as illustrated in FIG. 3A and as elsewhere described herein. Adonor DNA template for introducing the SEQ ID NO: 27 OgRRS into the 5′junction polynucleotide of an MIR162 locus includes the donor DNAtemplate of SEQ ID NO: 32. Integration of the SEQ ID NO: 32 donor DNAtemplate into the 5′ junction polynucleotide of an MIR162 locus canprovide an INIR12 locus comprising the CgRRS sequence set forth in SEQID NO: 37. Integration of the SEQ ID NO: 32 donor DNA template into the5′ junction polynucleotide of an MIR162 locus can provide an INIR12locus set forth in SEQ ID NO: 44 or 47, wherein the entirephosphomannose isomerase (pmi)-encoding selectable marker gene isretained. An INIR12 transgenic locus of SEQ ID NO: 47 comprising theCgRRS sequence set forth in SEQ ID NO: 37 in its 5′ junctionpolynucleotide is shown in FIG. 6. Integration of the SEQ ID NO: 32donor DNA template into the 5′ junction polynucleotide of an MIR162locus can provide an INIR12 locus set forth in SEQ ID NO: 45, whereinthe ZmUbiInt promoter and an operably linked phosphomannose isomerasecoding region of the pmi-encoding selectable marker gene are absent.

Such selectively excisable INIR12 transgenic loci can also comprisesignature protospacer adjacent motif (sPAM) sites and/or signature guideRNA recognition (sigRNAR) sites, wherein the sPAM and/or sigRNAR sitesare operably linked to both DNA junction polynucleotides of the INIR12transgenic locus. Such sigRNAR sites can be recognized by RdDe andsuitable guide RNAs containing crRNA complementary to heterologous DNAsequences adjacent to a PAM or sPAM site to provide for cleavage withinor near the two junction polynucleotides. Such heterologous sequenceswhich introduced at the sigRNAR site are at least 17 or 18 nucleotidesin length and are complementary to the crRNA of a guide RNA. In certainembodiments, the heterologous polynucleotide of the sigRNAR is about 17or 18 to about 24 nucleotides in length. Non-limiting features of theheterologous DNA sequences in the sigRNAR include: (i) absence ofsignificant homology or sequence identity (e.g., less than 50% sequenceidentity across the entire length of the heterologous sequence) to anyother endogenous or transgenic sequences present in the transgenic plantgenome or in other transgenic genomes of the maize plant being edited(ii) absence of significant homology or sequence identity (e.g., lessthan 50% sequence identity across the entire length of the heterologoussequence) of a heterologous sequence of a first sigRNAR site to aheterologous sequence of a second or third sigRNAR site; and/or (ii)optimization of the heterologous sequence for recognition by the RdDeand guide RNA when used in conjunction with a particular PAM sequence.In certain embodiments, the sigRNAR sites which are created arerecognized by the same class of RdDe (e.g., Class 2 type II or Class 2type V) or by the same RdDe (e.g., both sPAMs or PAMs of the sigRNARrecognized by the same RdDe (e.g., Cas9 or Cas 12 RdDe). In certainembodiments, the same sigRNAR sites can be introduced in both 5′ and 3′junction polynucleotides to permit excision of the INIR12 transgeniclocus by a single guide RNA and a single RdDe. In certain embodiments,different sets of distinct sigRNAR sites can be introduced in the 5′ and3′ junction polynucleotides of different transgenic loci to permitselective excision of any single transgenic locus by a single guide RNAand a single RdDe directed to the distinct sigRNAR sites that flank thetransgenic locus. A sigRNAR site can be created in the plant genome byinserting the heterologous sequence adjacent to a pre-existing PAMsequence using genome editing molecules. A sigRNAR site can be createdin the plant genome by inserting the heterologous sequence adjacent to apreexisting PAM sequence using genome editing molecules. A sigRNAR sitealso can be created in the plant genome by inserting both theheterologous sequence and an associated PAM or sPAM site in a junctionpolynucleotide. Such insertions can be made in non-transgenic plantgenomic DNA of the junction polynucleotide, in the inserted transgenicDNA of the junction polynucleotide, or can span the junction comprisingboth non-transgenic plant genomic DNA and inserted transgenic DNA of thejunction polynucleotide. Such nucleotide insertions can be effected inthe plant genome by using gene editing molecules (e.g., RdDe and guideRNAs, RNA dependent nickases and guide RNAs, Zinc Finger nucleases ornickases, or TALE nucleases or nickases) which introduce blunt doublestranded breaks or staggered double stranded breaks in the DNA junctionpolynucleotides. In the case of DNA insertions, the genome editingmolecules can also in certain embodiments further comprise a donor DNAtemplate or other DNA template which comprises the heterologousnucleotides for insertion. Guide RNAs can be directed to the junctionpolynucleotides by using a pre-existing PAM site located within oradjacent to a junction polynucleotide of the transgenic locus.

Also provided are unique transgenic locus excision sites created byexcision of INIR12 transgenic loci or selectively excisable INIR12transgenic loci, DNA molecules comprising the INIR12 transgenic loci orunique fragments thereof (i.e., fragments of an INIR12 locus which arenot found in an MIR162 transgenic locus), INIR12 plants comprising thesame, biological samples containing the DNA, nucleic acid markersadapted for detecting the DNA molecules, and related methods ofidentifying maize plants comprising unique INIR12 transgenic locusexcision sites and unique fragments of a INIR12 transgenic locus. DNAmolecules comprising unique fragments of an INIR12 transgenic locus arediagnostic for the presence of an INIR12 transgenic locus or fragmentsthereof in a maize plant, maize cell, maize seed, products obtainedtherefrom (e.g., seed meal or stover), and biological samples. DNAmolecules comprising unique fragments of an INIR12 transgenic locusinclude DNA molecules comprising modified 5′ junction polynucleotides.Unique 5′ junction polynucleotides of an INIR12 transgenic locusinclude: (i) a DNA molecule comprising nucleotides corresponding tonucleotides 1080 or 1082 to 1102 or 1104 of SEQ ID NO:1 or SEQ ID NO:46with the proviso that the DNA molecules is not identical to residues1080 or 1082 to 1102 or 1104 of SEQ ID NO:1 or SEQ ID NO:46); or (ii)any one of SEQ ID NO: 7, 8, or 9, with the proviso that the DNAmolecules is not identical to residues 1080 or 1082 to 1102 or 1104 ofSEQ ID NO:1 or SEQ ID NO:46; or (iii) or SEQ ID NO: 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 39, or 40. DNA molecules comprising unique fragmentsof an INIR12 transgenic locus also include DNA molecules comprisingmodified junction polynucleotides containing CgRRS sequences comprisinginsertions of OgRRS sequences (e.g., a CgRRS element comprising SEQ IDNO: 37). DNA molecules comprising unique fragments of an INIR12transgenic locus also include DNA molecules comprising deletionjunctions corresponding to residues spanning the deletion of thephosphomannose isomerase coding region and operably linked ZmUbiIntpromoter in the INIR12 transgenic locus. Such deletion junctions thuscomprise one or more nucleotides located between the 35S terminatorelement and the 5′ end of the ZmUbiInt promoter (e.g., nucleotides 5839to 5858 of SEQ ID NO:1 or SEQ ID NO:46) which are directly joined to(i.e., are contiguous with) nucleotides located between or at the 3′terminus of the pmi coding region and the 5′ end of the NOS terminatorin a MIR162 locus (e.g., nucleotides 9040 to 9105 of SEQ ID NO:1 or SEQID NO:46). Examples of unique INIR12 DNA fragment comprising a suchdeletion include nucleotides 5821 to 5850 of SEQ ID NO: 2, wherein oneor more nucleotides designated n are absent, independently selected froma guanine, a cytosine, an adenine residue, or a thymine residue,comprise or consist of 1 or more nucleotides corresponding tonucleotides 5831 to 5836 of SEQ ID NO:1 or SEQ ID NO:46 and/or compriseor consist of 1 or more nucleotides corresponding to nucleotides 9102 to9107 of SEQ ID NO:1 or SEQ ID NO:46 junction. Another example of aunique INIR12 DNA fragment comprising such a deletion junction includeSEQ ID NO: 25, which corresponds to residues 5821 to 5850 of an INIR12locus set forth in SEQ ID NO: 6. Another example of a unique INIR12 DNAfragment comprising such a deletion junction include SEQ ID NO: 41 and42. In certain embodiments, any of the aforementioned unique fragmentsof an INIR12 transgenic locus comprise DNA molecules of at least about18, 20, or 24 nucleotides to about 30, 50, 100, or 200 nucleotides inlength. Also provided herein are nucleic acid hybridization probes andprimers (e.g., for SNP analysis) adapted for detection of INIR12transgenic loci which can comprise all or part of any of theaforementioned DNA molecules and optionally a detectable label. Methodsand reagents (e.g., nucleic acid markers including nucleic acid probesand/or primers) for detecting plants, edited plant genomes, andbiological samples containing DNA molecules comprising the transgenicloci excision sites and/or non-essential DNA deletions are also providedherein. Detection of the DNA molecules can be achieved by anycombination of nucleic acid amplification (e.g., PCR amplification),hybridization, sequencing, and/or mass-spectrometry based techniques.Methods set forth for detecting junction nucleic acids in unmodifiedtransgenic loci set forth in US 20190136331 and U.S. Pat. No. 9,738,904,both incorporated herein by reference in their entireties, can beadapted for use in detection of the nucleic acids provided herein. Incertain embodiments, such detection is achieved by amplification and/orhybridization-based detection methods using a method (e.g., selectiveamplification primers) and/or probe (e.g., capable of selectivehybridization or generation of a specific primer extension product)which specifically recognizes the target DNA molecule (e.g., transgeniclocus excision site) but does not recognize DNA from an unmodifiedtransgenic locus. In certain embodiments, the hybridization probes cancomprise detectable labels (e.g., fluorescent, radioactive, epitope, andchemiluminescent labels). In certain embodiments, a single nucleotidepolymorphism detection assay can be adapted for detection of the targetDNA molecule (e.g., transgenic locus excision site). Detection of any ofthe aforementioned unique DNA fragments comprising SEQ ID NO: 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 39, 40, 41, and/or 42 in a biologicalsample indicates that the sample contains material from a INIR12 plantor seed.

Methods provided herein can be used to excise any transgenic locus wherethe first and second junction sequences comprising the endogenousnon-transgenic genomic DNA and the heterologous transgenic DNA which arejoined at the site of transgene insertion in the plant genome are knownor have been determined. In certain embodiments provided herein,transgenic loci can be removed from crop plant lines to obtain cropplant lines with tailored combinations of transgenic loci and optionallytargeted genetic changes. Such first and second junction sequences arereadily identified in new transgenic events by inverse PCR techniquesusing primers which are complementary the inserted transgenic sequences.In certain embodiments, the first and second junction sequences oftransgenic loci are published. An example of a transgenic locus whichcan be improved and used in the methods provided herein is the maizeMIR162 transgenic locus. The maize MIR162 transgenic locus and itstransgenic junction sequences are also depicted in FIG. 1. Maize plantscomprising the MIR162 transgenic locus and seed thereof have beencultivated, been placed in commerce, and have been described in avariety of publications by various governmental bodies. Databases whichhave compiled descriptions of the MIR162 transgenic locus include theInternational Service for the Acquisition of Agri-biotech Applications(ISAAA) database (available on the world wide web internet site“isaaa.org/gmapprovaldatabase/event”), the GenBit LLC database(available on the world wide web internet site“genbitgroup.com/en/gmo/gmodatabase”), and the Biosafety Clearing-House(BCH) database (available on the http internet site“bd.int/database/organisms”).

Sequences of the junction polynucleotides as well as the transgenicinsert(s) of an original MIR162 transgenic locus which can be improvedby the methods provided herein are set forth or otherwise provided inSEQ ID NO: 1, U.S. Pat. No. 8,455,720, the sequence of the MIR162 locusin the deposited seed of ATCC accession No. PTA-8166 (SEQ ID NO: 46),and elsewhere in this disclosure. In certain embodiments providedherein, the MIR162 transgenic locus set forth in SEQ ID NO: 1 or presentin the deposited seed of ATCC accession No. PTA-8166 (SEQ ID NO: 46) isreferred to as an original MIR162 transgenic locus. The MIR162transgenic locus set forth in SEQ ID NO:1 encodes the Vip3Aa19 protein.The MIR162 transgenic locus in the deposited seed of ATCC accession No.PTA-8166 (SEQ ID NO: 46) encodes the Vip3Aa20 protein. The Vip3Aa19 andVip3Aa20 proteins differ by one amino acid residue. The vip3Aa20 gene inSEQ ID NO: 46 encodes isoleucine at position 129 of the Vip3Aa20 proteinrather than the methionine residue at position 129 of the Vip3Aa19protein encoded by the vip3Aa19 gene of SEQ ID NO: 1. Allelic or othervariants of the sequence set forth in SEQ ID NO: 1 or SEQ ID NO:46, thepatent references set forth therein and incorporated herein by referencein their entireties, and elsewhere in this disclosure which may bepresent in certain variant MIR162 transgenic plant loci (e.g., progenyof deposited seed of accession No. PTA-8166 which contain allelicvariants of SEQ ID NO:1 or SEQ ID NO:46 or progeny originating fromtransgenic plant cells comprising the original MIR162 transgenic setforth in U.S. Pat. No. 8,455,720 which contain allelic variants of SEQID NO:1 or SEQ ID NO:46) can also be improved by identifying sequencesin the variants that correspond to the sequences of SEQ ID NO: 1 or SEQID NO: 46 by performing a pairwise alignment (e.g., using CLUSTAL O1.2.4 with default parameters) and making corresponding changes in theallelic or other variant sequences. Such allelic or other variantsequences include sequences having at least 85%, 90%, 95%, 98%, or 99%sequence identity across the entire length or at least 20, 40, 100, 500,1,000, 2,000, 4,000, 8,000, 10,000, or 10,579 nucleotides of SEQ ID NO:1 or SEQ ID NO: 46. Also provided are plants, plant parts includingseeds, genomic DNA, and/or DNA obtained from INIR12 plants whichcomprise one or more modifications (e.g., via insertion of a CgRRS in ajunction polynucleotide sequence) which provide for selective excisionof the INIR12 transgenic locus or a portion thereof (e.g., the Vip3Acoding region and operably linked promoter). Such INIR12 transgenic locican be treated with gene editing molecules (e.g., RdDe and gRNA(s)) toobtain plants wherein a segment comprising, consisting essentially of,or consisting of the INIR12 transgenic locus or a portion thereof (e.g.,the Vip3A coding region and operably linked promoter) is deleted. Incertain embodiments, the MIR162 transgenic loci set forth in SEQ ID NO:1or SEQ ID NO:46 and allelic variants thereof are further modified bydeletion of a segment of DNA comprising, consisting essentially of, orconsisting of a selectable marker gene or portions thereof (e.g, the pmicoding region and operably linked ZmUbi promoter) and/or non-essentialDNA (e.g., T-DNA border sequences or anything other than theZmUbi1::VIP3a::t35S expression cassette) to obtain INIR12 transgenicloci. In certain embodiments, the INIR12 transgenic locus comprises adeletion of the phosphomannose isomerase (PMI) coding region andoperably linked ZmUbi promoter which are in a MIR162 transgenic locus.Also provided herein are methods of detecting plants, genomic DNA,and/or DNA obtained from plants comprising a INIR12 transgenic locuswhich contains one or more of a CgRRS, deletions of selectable markergenes, deletions of non-essential DNA, and/or a transgenic locusexcision site. A first junction polynucleotide of a MIR162 transgeniclocus can comprise either one of the junction polynucleotides found atthe 5′ end or the 3′ end of any one of the sequences set forth in SEQ IDNO:1 or SEQ ID NO:46, allelic variants thereof, or other variantsthereof. An OgRRS can be found within non-transgenic DNA, transgenicDNA, or a combination thereof in either one of the junctionpolynucleotides of any one of SEQ ID NO:1 or SEQ ID NO:46, allelicvariants thereof, or other variants thereof. A second junctionpolynucleotide of a transgenic locus can comprise either one of thejunction polynucleotides found at the 5′ or 3′ end of any one of thesequences set forth in SEQ ID NO:1 or SEQ ID NO:46, allelic variantsthereof, or other variants thereof. A CgRRS can be introduced withintransgenic, non-transgenic DNA, or a combination thereof of either oneof the junction polynucleotides of any one of SEQ ID NO:1 or SEQ IDNO:46, allelic variants thereof, or other variants thereof to obtain anINIR12 transgenic locus. In certain embodiments, the OgRRS is found innon-transgenic DNA or transgenic DNA of the 5′ junction polynucleotideof a transgenic locus of any one of SEQ ID NO:1 or SEQ ID NO:46, allelicvariants thereof, or other variants thereof and the corresponding CgRRSis introduced into the transgenic DNA, non-transgenic DNA, or acombination thereof in the 3′ junction polynucleotide of the MIR162transgenic locus of SEQ ID NO: 1, SEQ ID NO: 46, allelic variantsthereof, or other variants thereof to obtain an INIR12 transgenic locus.In other embodiments, the OgRRS is found in non-transgenic DNA ortransgenic DNA of the 3′ junction polynucleotide of the MIR162transgenic locus of any one of SEQ ID NO: 1, SEQ ID NO: 46, allelicvariants thereof, or other variants thereof and the corresponding CgRRSis introduced into the transgenic DNA, non-transgenic DNA, or acombination thereof in the 5′ junction polynucleotide of the transgeniclocus of SEQ ID NO: 1, SEQ ID NO: 46, allelic variants thereof, or othervariants thereof to obtain an INIR12 transgenic locus.

In certain embodiments, the CgRRS is comprised in whole or in part of anexogenous DNA molecule that is introduced into a DNA junctionpolynucleotide by genome editing. In certain embodiments, the guide RNAhybridization site of the CgRRS is operably linked to a pre-existing PAMsite in the transgenic DNA or non-transgenic DNA of the transgenic plantgenome. In other embodiments, the guide RNA hybridization site of theCgRRS is operably linked to a new PAM site that is introduced in the DNAjunction polynucleotide by genome editing. A CgRRS can be located innon-transgenic plant genomic DNA of a DNA junction polynucleotide of anINIR12 transgenic locus, in transgenic DNA of a DNA junctionpolynucleotide of an INIR12 transgenic locus or can span the junction ofthe transgenic and non-transgenic DNA of a DNA junction polynucleotideof an INIR12 transgenic locus. An OgRRS can likewise be located innon-transgenic plant genomic DNA of a DNA junction polynucleotide of anINIR12 transgenic locus, in transgenic DNA of a DNA junctionpolynucleotide of an INIR12 transgenic locus or can span the junction ofthe transgenic and non-transgenic DNA of a DNA junction polynucleotideof an INIR12 transgenic locus.

Methods provided herein can be used in a variety of breeding schemes toobtain elite crop plants comprising subsets of desired modifiedtransgenic loci comprising an OgRRS and a CgRRS operably linked tojunction polynucleotide sequences and transgenic loci excision siteswhere undesired transgenic loci or portions thereof have been removed(e.g., by use of the OgRRS and a CgRRS). Such methods are useful atleast insofar as they allow for production of distinct useful donorplant lines each having unique sets of modified transgenic loci and, insome instances, targeted genetic changes that are tailored for distinctgeographies and/or product offerings. In an illustrative andnon-limiting example, a different product lines comprising transgenicloci conferring only two of three types of herbicide tolerance (e.g.,glyphosate, glufosinate, and dicamba) can be obtained from a singledonor line comprising three distinct transgenic loci conferringresistance to all three herbicides. In certain aspects, plantscomprising the subsets of undesired transgenic loci and transgenic lociexcision sites can further comprise targeted genetic changes. Such elitecrop plants can be inbred plant lines or can be hybrid plant lines. Incertain embodiments, at least two transgenic loci (e.g., transgenic lociincluding an INIR12 and another modified transgenic locus wherein anOgRRS and a CgRRS site is operably linked to a first and a secondjunction sequence and optionally a selectable marker gene and/ornon-essential DNA are deleted) are introgressed into a desired donorline comprising elite crop plant germplasm and then subjected to genomeediting molecules to recover plants comprising one of the twointrogressed transgenic loci as well as a transgenic loci excision siteintroduced by excision of the other transgenic locus or portion thereofby the genome editing molecules. In certain embodiments, the genomeediting molecules can be used to remove a transgenic locus and introducetargeted genetic changes in the crop plant genome. Introgression can beachieved by backcrossing plants comprising the transgenic loci to arecurrent parent comprising the desired elite germplasm and selectingprogeny with the transgenic loci and recurrent parent germplasm. Suchbackcrosses can be repeated and/or supplemented by molecular assistedbreeding techniques using SNP or other nucleic acid markers to selectfor recurrent parent germplasm until a desired recurrent parentpercentage is obtained (e.g., at least about 95%, 96%, 97%, 98%, or 99%recurrent parent percentage). A non-limiting, illustrative depiction ofa scheme for obtaining plants with both subsets of transgenic loci andthe targeted genetic changes is shown in the FIG. 2 (bottom“Alternative” panel), where two or more of the transgenic loci (“Event”in FIG. 2) are provided in Line A and then moved into elite crop plantgermplasm by introgression. In the non-limiting FIG. 2 illustration,introgression can be achieved by crossing a “Line A” comprising two ormore of the modified transgenic loci to the elite germplasm and thenbackcrossing progeny of the cross comprising the transgenic loci to theelite germplasm as the recurrent parent) to obtain a “Universal Donor”(e.g. Line A+ in FIG. 2) comprising two or more of the modifiedtransgenic loci. This elite germplasm containing the modified transgenicloci (e.g. “Universal Donor” of FIG. 2) can then be subjected to genomeediting molecules which can excise at least one of the transgenic loci(“Event Removal” in FIG. 2) and introduce other targeted genetic changes(“GE” in FIG. 2) in the genomes of the elite crop plants containing oneof the transgenic loci and a transgenic locus excision sitecorresponding to the removal site of one of the transgenic loci. Suchselective excision of transgenic loci or portion thereof can be effectedby contacting the genome of the plant comprising two transgenic lociwith gene editing molecules (e.g., RdDe and gRNAs, TALENS, and/or ZFN)which recognize one transgenic loci but not another transgenic loci.Genome editing molecules that provide for selective excision of a firstmodified transgenic locus comprising an OgRRS and a CgRRS include a gRNAthat hybridizes to the OgRRS and CgRRS of the first modified transgeniclocus and an RdDe that recognizes the gRNA/OgRRS and gRNA/CgRRScomplexes. Distinct plant lines with different subsets of transgenicloci and desired targeted genetic changes are thus recovered (e.g.,“Line B-1,” “Line B-2,” and “Line B-3” in FIG. 2). In certainembodiments, it is also desirable to bulk up populations of inbred elitecrop plants or their seed comprising the subset of transgenic loci and atransgenic locus excision site by selfing. In certain embodiments,inbred progeny of the selfed maize plants comprising the INIR12transgenic loci can be used as a pollen donor or recipient for hybridseed production. Such hybrid seed and the progeny grown therefrom cancomprise a subset of desired transgenic loci and a transgenic lociexcision site.

Hybrid plant lines comprising elite crop plant germplasm, at least onetransgenic locus and at least one transgenic locus excision site, and incertain aspects, additional targeted genetic changes are also providedherein. Methods for production of such hybrid seed can comprise crossingelite crop plant lines where at least one of the pollen donor orrecipient comprises at least the transgenic locus and a transgenic locusexcision site and/or additional targeted genetic changes. In certainembodiments, the pollen donor and recipient will comprise germplasm ofdistinct heterotic groups and provide hybrid seed and plants exhibitingheterosis. In certain embodiments, the pollen donor and recipient caneach comprise a distinct transgenic locus which confers either adistinct trait (e.g., herbicide tolerance or insect resistance), adifferent type of trait (e.g., tolerance to distinct herbicides or todistinct insects such as coleopteran or lepidopteran insects), or adifferent mode-of-action for the same trait (e.g., resistance tocoleopteran insects by two distinct modes-of-action or resistance tolepidopteran insects by two distinct modes-of-action). In certainembodiments, the pollen recipient will be rendered male sterile orconditionally male sterile. Methods for inducing male sterility orconditional male sterility include emasculation (e.g., detasseling),cytoplasmic male sterility, chemical hybridizing agents or systems, atransgenes or transgene systems, and/or mutation(s) in one or moreendogenous plant genes. Descriptions of various male sterility systemsthat can be adapted for use with the elite crop plants provided hereinare described in Wan et al. Molecular Plant; 12, 3, (2019):321-342 aswell as in U.S. Pat. No. 8,618,358; US 20130031674; and US 2003188347.

In certain embodiments, it will be desirable to use genome editingmolecules to make modified transgenic loci by introducing a CgRRS intothe transgenic loci, to excise modified transgenic loci comprising anOgRRS and a CgRRS, and/or to make targeted genetic changes in elite cropplant or other germplasm. Techniques for effecting genome editing incrop plants (e.g., maize,) include use of morphogenic factors such asWuschel (WUS), Ovule Development Protein (ODP), and/or Babyboom (BBM)which can improve the efficiency of recovering plants with desiredgenome edits. In some aspects, the morphogenic factor comprises WUS1,WUS2, WUS3, WOX2A, WOX4, WOX5, WOX9, BBM2, BMN2, BMN3, and/or ODP2. Incertain embodiments, compositions and methods for using WUS, BBM, and/orODP, as well as other techniques which can be adapted for effectinggenome edits in elite crop plant and other germplasm, are set forth inUS 20030082813, US 20080134353, US 20090328252, US 20100100981, US20110165679, US 20140157453, US 20140173775, and US 20170240911, whichare each incorporated by reference in their entireties. In certainembodiments, the genome edits can be effected in regenerable plant parts(e.g.; plant embryos) of elite crop plants by transient provision ofgene editing molecules or polynucleotides encoding the same and do notnecessarily require incorporating a selectable marker gene into theplant genome (e.g., US 20160208271 and US 20180273960, both incorporatedherein by reference in their entireties; Svitashev et al. Nat Commun.2016; 7:13274).

In certain embodiments, edited transgenic plant genomes, transgenicplant cells, parts, or plants containing those genomes, and DNAmolecules obtained therefrom, can comprise a desired subset oftransgenic loci and/or comprise at least one transgenic locus excisionsite. In certain embodiments, a segment comprising an INIR12 transgeniclocus comprising an OgRRS in non-transgenic DNA of a 1^(st) junctionpolynucleotide sequence and a CgRRS in a 2^(nd) junction polynucleotidesequence is deleted with a gRNA and RdDe that recognize the OgRRS andthe CgRRS to produce an INIR12 transgenic locus excision site. Incertain embodiments, a segment comprising an INIR12 transgenic locuscomprising a sPAM and/or a sigRNAR site in a 1^(st) junctionpolynucleotide sequence and a sPAM and/or a sigRNAR in a 2^(nd) junctionpolynucleotide sequence is deleted with at least one gRNA and RdDe thatrecognize the sPAM and/or a sigRNAR to produce an INIR12 transgeniclocus excision site. In certain embodiments, the transgenic locusexcision site can comprise a contiguous segment of DNA comprising atleast 10 base pairs of DNA that is telomere proximal to the deletedsegment of the transgenic locus and at least 10 base pairs of DNA thatis centromere proximal to the deleted segment of the transgenic locuswherein the transgenic DNA (i.e., the heterologous DNA) that has beeninserted into the crop plant genome has been deleted. In certainembodiments where a segment comprising a transgenic locus has beendeleted, the transgenic locus excision site can comprise a contiguoussegment of DNA comprising at least 10 base pairs DNA that is telomereproximal to the deleted segment of the transgenic locus and at least 10base pairs of DNA that is centromere proximal DNA to the deleted segmentof the transgenic locus wherein the heterologous transgenic DNA and atleast 1, 2, 5, 10, 20, 50, or more base pairs of endogenous DNA locatedin a 5′ junction sequence and/or in a 3′ junction sequence of theoriginal transgenic locus that has been deleted. In such embodimentswhere DNA comprising the transgenic locus is deleted, a transgenic locusexcision site can comprise at least 10 base pairs of DNA that istelomere proximal to the deleted segment of the transgenic locus and atleast 10 base pairs of DNA that is centromere proximal to the deletedsegment of the transgenic locus wherein all of the transgenic DNA isabsent and either all or less than all of the endogenous DNA flankingthe transgenic DNA sequences are present. In certain embodiments where asegment consisting essentially of an original transgenic locus has beendeleted, the transgenic locus excision site can be a contiguous segmentof at least 10 base pairs of DNA that is telomere proximal to thedeleted segment of the transgenic locus and at least 10 base pairs ofDNA that is centromere proximal to the deleted segment of the transgeniclocus wherein less than all of the heterologous transgenic DNA that hasbeen inserted into the crop plant genome is excised. In certainaforementioned embodiments where a segment consisting essentially of anoriginal transgenic locus has been deleted, the transgenic locusexcision site can thus contain at least 1 base pair of DNA or 1 to about2 or 5, 8, 10, 20, or 50 base pairs of DNA comprising the telomereproximal and/or centromere proximal heterologous transgenic DNA that hasbeen inserted into the crop plant genome. In certain embodiments where asegment consisting of an original transgenic locus has been deleted, thetransgenic locus excision site can contain a contiguous segment of DNAcomprising at least 10 base pairs of DNA that is telomere proximal tothe deleted segment of the transgenic locus and at least 10 base pairsof DNA that is centromere proximal to the deleted segment of thetransgenic locus wherein the heterologous transgenic DNA that has beeninserted into the crop plant genome is deleted. In certain embodimentswhere DNA consisting of the transgenic locus is deleted, a transgeniclocus excision site can comprise at least 10 base pairs of DNA that istelomere proximal to the deleted segment of the transgenic locus and atleast 10 base pairs of DNA that is centromere proximal to the deletedsegment of the transgenic locus wherein all of the heterologoustransgenic DNA that has been inserted into the crop plant genome isdeleted and all of the endogenous DNA flanking the heterologoussequences of the transgenic locus is present. In any of theaforementioned embodiments or in other embodiments, the continuoussegment of DNA comprising the transgenic locus excision site can furthercomprise an insertion of 1 to about 2, 5, 10, 20, or more nucleotidesbetween the DNA that is telomere proximal to the deleted segment of thetransgenic locus and the DNA that is centromere proximal to the deletedsegment of the transgenic locus. Such insertions can result either fromendogenous DNA repair and/or recombination activities at the doublestranded breaks introduced at the excision site and/or from deliberateinsertion of an oligonucleotide. Plants, edited plant genomes,biological samples, and DNA molecules (e.g., including isolated orpurified DNA molecules) comprising the INIR12 transgenic loci excisionsites are provided herein.

In other embodiments, a segment comprising a INIR12 transgenic locus(e.g., a transgenic locus comprising an OgRRS in non-transgenic DNA of a1^(st) junction sequence and a CgRRS in a 2^(nd) junction sequence) canbe deleted with a gRNA and RdDe that recognize the OgRRS and the CgRRSand replaced with DNA comprising the endogenous non-transgenic plantgenomic DNA present in the genome prior to transgene insertion. Anon-limiting example of such replacements can be visualized in FIG. 3C,where the donor DNA template can comprise the endogenous non-transgenicplant genomic DNA present in the genome prior to transgene insertionalong with sufficient homology to non-transgenic DNA on each side of theexcision site to permit homology-directed repair. In certainembodiments, the endogenous non-transgenic plant genomic DNA present inthe genome prior to transgene insertion can be at least partiallyrestored. In certain embodiments, the endogenous non-transgenic plantgenomic DNA present in the genome prior to transgene insertion can beessentially restored such that no more than about 5, 10, or 20 to about50, 80, or 100 nucleotides are changed relative to the endogenous DNA atthe essentially restored excision site.

In certain embodiments, edited transgenic plant genomes and transgenicplant cells, plant parts, or plants containing those edited genomes,comprising a modification of an original transgenic locus, where themodification comprises an OgRRS and a CgRRS which are operably linked toa 1^(st) and a 2^(nd) junction sequence, respectively or irrespectively,and optionally further comprise a deletion of a segment of the originaltransgenic locus. In certain embodiments, the modification comprises twoor more separate deletions and/or there is a modification in two or moreoriginal transgenic plant loci. In certain embodiments, the deletedsegment comprises, consists essentially of, or consists of a segment ofnon-essential DNA in the transgenic locus. Illustrative examples ofnon-essential DNA include but are not limited to synthetic cloning sitesequences, duplications of transgene sequences; fragments of transgenesequences, and Agrobacterium right and/or left border sequences. Incertain embodiments, the non-essential DNA is a duplication and/orfragment of a promoter sequence and/or is not the promoter sequenceoperably linked in the cassette to drive expression of a transgene. Incertain embodiments, excision of the non-essential DNA improves acharacteristic, functionality, and/or expression of a transgene of thetransgenic locus or otherwise confers a recognized improvement in atransgenic plant comprising the edited transgenic plant genome. Incertain embodiments, the non-essential DNA does not comprise DNAencoding a selectable marker gene. In certain embodiments of an editedtransgenic plant genome, the modification comprises a deletion of thenon-essential DNA and a deletion of a selectable marker gene. Themodification producing the edited transgenic plant genome could occur byexcising both the non-essential DNA and the selectable marker gene atthe same time, e.g., in the same modification step, or the modificationcould occur step-wise. For example, an edited transgenic plant genome inwhich a selectable marker gene has previously been removed from thetransgenic locus can comprise an original transgenic locus from which anon-essential DNA is further excised and vice versa. In certainembodiments, the modification comprising deletion of the non-essentialDNA and deletion of the selectable marker gene comprises excising asingle segment of the original transgenic locus that comprises both thenon-essential DNA and the selectable marker gene. Such modificationwould result in one excision site in the edited transgenic genomecorresponding to the deletion of both the non-essential DNA and theselectable marker gene. In certain embodiments, the modificationcomprising deletion of the non-essential DNA and deletion of theselectable marker gene comprises excising two or more segments of theoriginal transgenic locus to achieve deletion of both the non-essentialDNA and the selectable marker gene. Such modification would result in atleast two excision sites in the edited transgenic genome correspondingto the deletion of both the non-essential DNA and the selectable markergene. In certain embodiments of an edited transgenic plant genome, priorto excision, the segment to be deleted is flanked by operably linkedprotospacer adjacent motif (PAM) sites in the original or unmodifiedtransgenic locus and/or the segment to be deleted encompasses anoperably linked PAM site in the original or unmodified transgenic locus.In certain embodiments, following excision of the segment, the resultingedited transgenic plant genome comprises PAM sites flanking the deletionsite in the modified transgenic locus. In certain embodiments of anedited transgenic plant genome, the modification comprises amodification of a MIR162 transgenic locus.

In certain embodiments, improvements in a transgenic plant locus areobtained by introducing a new cognate guide RNA recognition site (CgRRS)which is operably linked to a DNA junction polynucleotide of thetransgenic locus in the transgenic plant genome. Such CgRRS sites can berecognized by RdDe and a single suitable guide RNA directed to the CgRRSand the originator gRNA Recognition Site (OgRRS) to provide for cleavagewithin the junction polynucleotides which flank an INIR12 transgeniclocus. In certain embodiments, the CgRRS/gRNA and OgRRS/gRNAhybridization complexes are recognized by the same class of RdDe (e.g.,Class 2 type II or Class 2 type V) or by the same RdDe (e.g., both theCgRRS/gRNA and OgRRS/gRNA hybridization complexes recognized by the sameCas9 or Cas 12 RdDe). Such CgRRS and OgRRS can be recognized by RdDe andsuitable guide RNAs containing crRNA sufficiently complementary to theguide RNA hybridization site DNA sequences adjacent to the PAM site ofthe CgRRS and the OgRRS to provide for cleavage within or near the twojunction polynucleotides. Suitable guide RNAs can be in the form of asingle gRNA comprising a crRNA or in the form of a crRNA/tracrRNAcomplex. In the case of the OgRRS site, the PAM and guide RNAhybridization site are endogenous DNA polynucleotide molecules found inthe plant genome. In certain embodiments where the CgRRS is introducedinto the plant genome by genome editing, gRNA hybridization sitepolynucleotides introduced at the CgRRS are at least 17 or 18nucleotides in length and are complementary to the crRNA of a guide RNA.In certain embodiments, the gRNA hybridization site sequence of theOgRRS and/or the CgRRS is about 17 or 18 to about 24 nucleotides inlength. The gRNA hybridization site sequence of the OgRRS and the gRNAhybridization site of the CgRRS can be of different lengths or comprisedifferent sequences so long as there is sufficient complementarity topermit hybridization by a single gRNA and recognition by a RdDe thatrecognizes and cleaves DNA at the gRNA/OgRRS and gRNA/CgRRS complex. Incertain embodiments, the guide RNA hybridization site of the CgRRScomprise about a 17 or 18 to about 24 nucleotide sequence which isidentical to the guide RNA hybridization site of the OgRRS. In otherembodiments, the guide RNA hybridization site of the CgRRS compriseabout a 17 or 18 to about 24 nucleotide sequence which has one, two,three, four, or five nucleotide insertions, deletions or substitutionswhen compared to the guide RNA hybridization site of the OgRRS. CertainCgRRS comprising a gRNA hybridization site containing has one, two,three, four, or five nucleotide insertions, deletions or substitutionswhen compared to the guide RNA hybridization site of the OgRRS canundergo hybridization with a gRNA which is complementary to the OgRRSgRNA hybridization site and be cleaved by certain RdDe. Examples ofmismatches between gRNAs and guide RNA hybridization sites which allowfor RdDe recognition and cleavage include mismatches resulting from bothnucleotide insertions and deletions in the DNA which is hybridized tothe gRNA (e.g., Lin et al., doi: 10.1093/nar/gku402). In certainembodiments, an operably linked PAM site is co-introduced with the gRNAhybridization site polynucleotide at the CgRRS. In certain embodiments,the gRNA hybridization site polynucleotides are introduced at a positionadjacent to a resident endogenous PAM sequence in the junctionpolynucleotide sequence to form a CgRRS where the gRNA hybridizationsite polynucleotides are operably linked to the endogenous PAM site. Incertain embodiments, non-limiting features of the OgRRS, CgRRS, and/orthe gRNA hybridization site polynucleotides thereof include: (i) absenceof significant homology or sequence identity (e.g., less than 50%sequence identity across the entire length of the OgRRS, CgRRS, and/orthe gRNA hybridization site sequence) to any other endogenous ortransgenic sequences present in the transgenic plant genome or in othertransgenic genomes of the maize plant being transformed and edited; (ii)absence of significant homology or sequence identity (e.g., less than50% sequence identity across the entire length of the sequence) of asequence of a first OgRRS and a first CgRRS to a second OgRRS and asecond CgRRS which are operably linked to junction polynucleotides of adistinct transgenic locus; (iii) the presence of some sequence identity(e.g., about 25%, 40%, or 50% to about 60%, 70%, or 80%) between theOgRRS sequence and endogenous sequences present at the site where theCgRRS sequence is introduced; and/or (iv) optimization of the gRNAhybridization site polynucleotides for recognition by the RdDe and guideRNA when used in conjunction with a particular PAM sequence. In certainembodiments, the first and second OgRRS as well as the first and secondCgRRS are recognized by the same class of RdDe (e.g., Class 2 type II orClass 2 type V) or by the same RdDe (e.g., Cas9 or Cas 12 RdDe). Incertain embodiments, the first OgRRS site in a first junctionpolynucleotide and the CgRRS introduced in the second junctionpolynucleotide to permit excision of a first transgenic locus by a firstsingle guide RNA and a single RdDe. Such nucleotide insertions or genomeedits used to introduce CgRRS in a transgenic plant genome can beeffected in the plant genome by using gene editing molecules (e.g., RdDeand guide RNAs, RNA dependent nickases and guide RNAs, Zinc Fingernucleases or nickases, or TALE nucleases or nickases) which introduceblunt double stranded breaks or staggered double stranded breaks in theDNA junction polynucleotides. In the case of DNA insertions, the genomeediting molecules can also in certain embodiments further comprise adonor DNA template or other DNA template which comprises theheterologous nucleotides for insertion to form the CgRRS. Guide RNAs canbe directed to the junction polynucleotides by using a pre-existing PAMsite located within or adjacent to a junction polynucleotide of thetransgenic locus. Non-limiting examples of such pre-existing PAM sitespresent in junction polynucleotides, which can be used either inconjunction with an inserted heterologous sequence to form a CgRRS orwhich can be used to create a double stranded break to insert or createa CgRRS, include PAM sites recognized by a Cas12a enzyme. Non-limitingexamples where a CgRRS are created in a DNA sequence are illustrated inExample 2.

Transgenic loci comprising OgRRS and CgRRS in a first and a secondjunction polynucleotides can be excised from the genomes of transgenicplants by contacting the transgenic loci with RdDe or RNA directednickases, and a suitable guide RNA directed to the OgRRS and CgRRS. Anon-limiting example where a modified transgenic locus is excised from aplant genome by use of a gRNA and an RdDe that recognizes an OgRRS/gRNAand a CgRRS/gRNA complex and introduces dsDNA breaks in both junctionpolynucleotides and repaired by NHEJ is depicted in FIG. 3B. In thedepicted example set forth in FIG. 3B, the OgRRS site and the CgRRS siteare absent from the plant chromosome comprising the transgene excisionsite that results from the process. In other embodiments provided hereinwhere a modified transgenic locus is excised from a plant genome by useof a gRNA and an RdDe that recognizes an OgRRS/gRNA and a CgRRS/gRNAcomplex and repaired by NHEJ or microhomology-mediated end joining(MMEJ), the OgRRS and/or other non-transgenic sequences that wereoriginally present prior to transgene insertion are at least partiallyor essentially restored.

In certain embodiments, edited transgenic plant genomes provided hereincan lack one or more selectable and/or scoreable markers found in anoriginal event (transgenic locus). Original MIR162 transgenic loci(events), including those set forth in SEQ ID NO: 1), U.S. Pat. No.8,455,720, the sequence of the MIR162 locus in the deposited seed ofaccession No. PTA-8166 (SEQ ID NO: 46) and progeny thereof, contain aselectable phosphomannose isomerase (pmi) transgene marker conferring anability to grow on mannose. Transgenes encoding a phosphomannoseisomerase (pmi) can confer the ability to grow on mannose. In certainembodiments provided herein, the DNA element comprising, consistingessentially of, or consisting of the ZmUbi promoter which is operablylinked to a pmi coding region of an MIR162 transgenic locus is absentfrom an INIR12 transgenic locus. or scoreable marker transgenes can beexcised from an original transgenic locus by contacting the transgeniclocus with one or more gene editing molecules which introduce doublestranded breaks in the transgenic locus at the 5′ and 3′ end of theexpression cassette comprising the selectable marker transgene (e.g., anRdDe and guide RNAs directed to PAM sites located at the 5′ and 3′ endof the expression cassette comprising the selectable marker transgenes)and selecting for plant cells, plant parts, or plants wherein theselectable or scoreable marker has been excised. In certain embodiments,the selectable or scoreable marker transgene can be inactivated.Inactivation can be achieved by modifications including insertion,deletion, and/or substitution of one or more nucleotides in a promoterelement, 5′ or 3′ untranslated region (UTRs), intron, coding region,and/or 3′ terminator and/or polyadenylation site of the selectablemarker transgene. Such modifications can inactivate the selectable orscoreable marker transgene by eliminating or reducing promoter activity,introducing a missense mutation, and/or introducing a pre-mature stopcodon. In certain embodiments, the selectable and/or scoreable markertransgene can be replaced by an introduced transgene. In certainembodiments, an original transgenic locus that was contacted with geneediting molecules which introduce double stranded breaks in thetransgenic locus at the 5′ and 3′ end of the expression cassettecomprising the selectable marker and/or scoreable transgene can also becontacted with a suitable donor DNA template comprising an expressioncassette flanked by DNA homologous to remaining DNA in the transgeniclocus located 5′ and 3′ to the selectable marker excision site. Incertain embodiments, a coding region of the selectable and/or scoreablemarker transgene can be replaced with another coding region such thatthe replacement coding region is operably linked to the promoter and 3′terminator or polyadenylation site of the selectable and/or scoreablemarker transgene.

In certain embodiments, edited transgenic plant genomes provided hereincan comprise additional new introduced transgenes (e.g., expressioncassettes) inserted into the transgenic locus of a given event.Introduced transgenes inserted at the transgenic locus of an eventsubsequent to the event's original isolation can be obtained by inducinga double stranded break at a site within an original transgenic locus(e.g., with genome editing molecules including an RdDe and suitableguide RNA(s); a suitable engineered zinc-finger nuclease; a TALENprotein and the like) and providing an exogenous transgene in a donorDNA template which can be integrated at the site of the double strandedbreak (e.g. by homology-directed repair (HDR) or by non-homologousend-joining (NHEJ)). In certain embodiments, an OgRRS and a CgRRSlocated in a 1^(st) junction polynucleotide and a 2^(nd) junctionpolynucleotide, respectively, can be used to delete the transgenic locusand replace it with one or more new expression cassettes. In certainembodiments, such deletions and replacements are effected by introducingdsDNA breaks in both junction polynucleotides and providing the newexpression cassettes on a donor DNA template (e.g., in FIG. 3C, thedonor DNA template can comprise an expression cassette flanked by DNAhomologous to non-transgenic DNA located telomere proximal andcentromere proximal to the excision site). Suitable expression cassettesfor insertion include DNA molecules comprising promoters which areoperably linked to DNA encoding proteins and/or RNA molecules whichconfer useful traits which are in turn operably linked topolyadenylation sites or terminator elements. In certain embodiments,such expression cassettes can also comprise 5′ UTRs, 3′ UTRs, and/orintrons. Useful traits include biotic stress tolerance (e.g., insectresistance, nematode resistance, or disease resistance), abiotic stresstolerance (e.g., heat, cold, drought, and/or salt tolerance), herbicidetolerance, and quality traits (e.g., improved fatty acid compositions,protein content, starch content, and the like). Suitable expressioncassettes for insertion include expression cassettes which confer insectresistance, herbicide tolerance, biofuel use, or male sterility traitscontained in any of the transgenic events set forth in US PatentApplication Public. Nos. 20090038026, 20130031674, 20150361446,20170088904, 20150267221, 201662346688, and 20200190533 as well as inU.S. Pat. Nos. 6,342,660, 7,323,556, 8,575,434, 6,040,497, 8,759,618,7,157,281, 6,852,915, 7,705,216, 10,316,330, 8,618,358, 8,450,561,8,212,113, 9,428,765, 7,897,748, 8,273,959, 8,093,453, 8,901,378,9,994,863, 7,928,296, and 8,466,346, each of which are incorporatedherein by reference in their entireties.

In certain embodiments, INIR12 plants provided herein, including plantswith one or more transgenic loci, modified transgenic loci, and/orcomprising transgenic loci excision sites can further comprise one ormore targeted genetic changes introduced by one or more of gene editingmolecules or systems. Also provided are methods where the targetedgenetic changes are introduced and one or more transgenic loci areremoved from plants either in series or in parallel (e.g., as set forthin the non-limiting illustration in FIG. 2, bottom “Alternative” panel,where “GE” can represent targeted genetic changes induced by geneediting molecules and “Event Removal” represents excision of one or moretransgenic loci with gene editing molecules). Such targeted geneticchanges include those conferring traits such as improved yield, improvedfood and/or feed characteristics (e.g., improved oil, starch, protein,or amino acid quality or quantity), improved nitrogen use efficiency,improved biofuel use characteristics (e.g., improved ethanolproduction), male sterility/conditional male sterility systems (e.g., bytargeting endogenous MS26, MS45 and MSCA1 genes), herbicide tolerance(e.g., by targeting endogenous ALS, EPSPS, HPPD, or other herbicidetarget genes), delayed flowering, non-flowering, increased biotic stressresistance (e.g., resistance to insect, nematode, bacterial, or fungaldamage), increased abiotic stress resistance (e.g., resistance todrought, cold, heat, metal, or salt), enhanced lodging resistance,enhanced growth rate, enhanced biomass, enhanced tillering, enhancedbranching, delayed flowering time, delayed senescence, increased flowernumber, improved architecture for high density planting, improvedphotosynthesis, increased root mass, increased cell number, improvedseedling vigor, improved seedling size, increased rate of cell division,improved metabolic efficiency, and increased meristem size in comparisonto a control plant lacking the targeted genetic change. Types oftargeted genetic changes that can be introduced include insertions,deletions, and substitutions of one or more nucleotides in the cropplant genome. Sites in endogenous plant genes for the targeted geneticchanges include promoter, coding, and non-coding regions (e.g., 5′ UTRs,introns, splice donor and acceptor sites and 3′ UTRs). In certainembodiments, the targeted genetic change comprises an insertion of aregulatory or other DNA sequence in an endogenous plant gene.Non-limiting examples of regulatory sequences which can be inserted intoendogenous plant genes with gene editing molecules to effect targetedgenetic changes which confer useful phenotypes include those set forthin US Patent Application Publication 20190352655, which is incorporatedherein by example, such as: (a) auxin response element (AuxRE) sequence;(b) at least one D1-4 sequence (Ulmasov et al. (1997) Plant Cell,9:1963-1971), (c) at least one DR5 sequence (Ulmasov et al. (1997) PlantCell, 9:1963-1971); (d) at least one m5-DR5 sequence (Ulmasov et al.(1997) Plant Cell, 9:1963-1971); (e) at least one P3 sequence; (f) asmall RNA recognition site sequence bound by a corresponding small RNA(e.g., an siRNA, a microRNA (miRNA), a trans-acting siRNA as describedin U.S. Pat. No. 8,030,473, or a phased sRNA as described in U.S. Pat.No. 8,404,928; both of these cited patents are incorporated by referenceherein); (g) a microRNA (miRNA) recognition site sequence; (h) thesequence recognizable by a specific binding agent includes a microRNA(miRNA) recognition sequence for an engineered miRNA wherein thespecific binding agent is the corresponding engineered mature miRNA; (i)a transposon recognition sequence; (j) a sequence recognized by anethylene-responsive element binding-factor-associated amphiphilicrepression (EAR) motif; (k) a splice site sequence (e.g., a donor site,a branching site, or an acceptor site; see, for example, the splicesites and splicing signals set forth in the internet sitelemur[dot]amu[dot]edu[dot]pl/share/ERISdb/home.html); (l) a recombinaserecognition site sequence that is recognized by a site-specificrecombinase; (m) a sequence encoding an RNA or amino acid aptamer or anRNA riboswitch, the specific binding agent is the corresponding ligand,and the change in expression is upregulation or downregulation; (n) ahormone responsive element recognized by a nuclear receptor or ahormone-binding domain thereof; (o) a transcription factor bindingsequence; and (p) a polycomb response element (see Xiao et al. (2017)Nature Genetics, 49:1546-1552, doi: 10.1038/ng.3937). Non limitingexamples of target maize genes that can be subjected to targeted geneedits to confer useful traits include: (a) ZmIPK1 (herbicide tolerantand phytate reduced maize; Shukla et al., Nature. 2009; 459:437-41); (b)ZmGL2 (reduced epicuticular wax in leaves; Char et al. Plant BiotechnolJ. 2015; 13:1002); (c) ZmMTL (induction of haploid plants; Kelliher etal. Nature. 2017; 542:105); (d) Wx1 (high amylopectin content; US20190032070; incorporated herein by reference in its entirety); (e) TMS5(thermosensitive male sterile; Li et al. J Genet Genomics. 2017;44:465-8); (f) ALS (herbicide tolerance; Svitashev et al.; PlantPhysiol. 2015; 169:931-45); and (g) ARGOS8 (drought stress tolerance;Shi et al., Plant Biotechnol J. 2017; 15:207-16). Non-limiting examplesof target genes in crop plants including maize which can be subjected totargeted genetic changes which confer useful phenotypes include thoseset forth in US Patent Application Nos. 20190352655, 20200199609,20200157554, and 20200231982, which are each incorporated herein intheir entireties; and Zhang et al. (Genome Biol. 2018; 19: 210).

Gene editing molecules of use in methods provided herein includemolecules capable of introducing a double-strand break (“DSB”) orsingle-strand break (“SSB”) in double-stranded DNA, such as in genomicDNA or in a target gene located within the genomic DNA as well asaccompanying guide RNA or donor DNA template polynucleotides. Examplesof such gene editing molecules include: (a) a nuclease comprising anRNA-guided nuclease, an RNA-guided DNA endonuclease or RNA directed DNAendonuclease (RdDe), a class 1 CRISPR type nuclease system, a type IICas nuclease, a Cas9, a nCas9 nickase, a type V Cas nuclease, a Cas12anuclease, a nCas12a nickase, a Cas12d (CasY), a Cas12e (CasX), a Cas12b(C2c1), a Cas12c (C2c3), a Cas12i, a Cas12j, a Cas14, an engineerednuclease, a codon-optimized nuclease, a zinc-finger nuclease (ZFN) ornickase, a transcription activator-like effector nuclease (TAL-effectornuclease or TALEN) or nickase (TALE-nickase), an Argonaute, and ameganuclease or engineered meganuclease; (b) a polynucleotide encodingone or more nucleases capable of effectuating site-specific alteration(including introduction of a DSB or SSB) of a target nucleotidesequence; (c) a guide RNA (gRNA) for an RNA-guided nuclease, or a DNAencoding a gRNA for an RNA-guided nuclease; (d) donor DNA templatepolynucleotides; and (e) other DNA templates (dsDNA, ssDNA, orcombinations thereof) suitable for insertion at a break in genomic DNA(e.g., by non-homologous end joining (NHEJ) or microhomology-mediatedend joining (MMEJ).

CRISPR-type genome editing can be adapted for use in the plant cells andmethods provided herein in several ways. CRISPR elements, e.g., geneediting molecules comprising CRISPR endonucleases and CRISPR guide RNAsincluding single guide RNAs or guide RNAs in combination with tracrRNAsor scoutRNA, or polynucleotides encoding the same, are useful ineffectuating genome editing without remnants of the CRISPR elements orselective genetic markers occurring in progeny. In certain embodiments,the CRISPR elements are provided directly to the eukaryotic cell (e.g.,plant cells), systems, methods, and compositions as isolated molecules,as isolated or semi-purified products of a cell free synthetic process(e.g., in vitro translation), or as isolated or semi-purified productsof in a cell-based synthetic process (e.g., such as in a bacterial orother cell lysate). In certain embodiments, genome-inserted CRISPRelements are useful in plant lines adapted for use in the methodsprovide herein. In certain embodiments, plants or plant cells used inthe systems, methods, and compositions provided herein can comprise atransgene that expresses a CRISPR endonuclease (e.g., a Cas9, aCpf1-type or other CRISPR endonuclease). In certain embodiments, one ormore CRISPR endonucleases with unique PAM recognition sites can be used.Guide RNAs (sgRNAs or crRNAs and a tracrRNA) to form an RNA-guidedendonuclease/guide RNA complex which can specifically bind sequences inthe gDNA target site that are adjacent to a protospacer adjacent motif(PAM) sequence. The type of RNA-guided endonuclease typically informsthe location of suitable PAM sites and design of crRNAs or sgRNAs.G-rich PAM sites, e.g., 5′-NGG are typically targeted for design ofcrRNAs or sgRNAs used with Cas9 proteins. Examples of PAM sequencesinclude 5′-NGG (Streptococcus pyogenes), 5′-NNAGAA (Streptococcusthermophilus CRISPR1), 5′-NGGNG (Streptococcus thermophilus CRISPR3),5′-NNGRRT or 5′-NNGRR (Staphylococcus aureus Cas9, SaCas9), and5′-NNNGATT (Neisseria meningitidis). T-rich PAM sites (e.g., 5′-TTN or5′-TTTV, where “V” is A, C, or G) are typically targeted for design ofcrRNAs or sgRNAs used with Cas12a proteins. In some instances, Cas12acan also recognize a 5′-CTA PAM motif. Other examples of potentialCas12a PAM sequences include TTN, CTN, TCN, CCN, TTTN, TCTN, TTCN, CTTN,ATTN, TCCN, TTGN, GTTN, CCCN, CCTN, TTAN, TCGN, CTCN, ACTN, GCTN, TCAN,GCCN, and CCGN (wherein N is defined as any nucleotide). Cpf1 (i.e.,Cas12a) endonuclease and corresponding guide RNAs and PAM sites aredisclosed in US Patent Application Publication 2016/0208243 A1, which isincorporated herein by reference for its disclosure of DNA encoding Cpf1endonucleases and guide RNAs and PAM sites. Introduction of one or moreof a wide variety of CRISPR guide RNAs that interact with CRISPRendonucleases integrated into a plant genome or otherwise provided to aplant is useful for genetic editing for providing desired phenotypes ortraits, for trait screening, or for gene editing mediated traitintrogression (e.g., for introducing a trait into a new genotype withoutbackcrossing to a recurrent parent or with limited backcrossing to arecurrent parent). Multiple endonucleases can be provided in expressioncassettes with the appropriate promoters to allow multiple genome siteediting.

CRISPR technology for editing the genes of eukaryotes is disclosed in USPatent Application Publications 2016/0138008A1 and US2015/0344912A1, andin U.S. Pat. Nos. 8,697,359, 8,771,945, 8,945,839, 8,999,641, 8,993,233,8,895,308, 8,865,406, 8,889,418, 8,871,445, 8,889,356, 8,932,814,8,795,965, and 8,906,616. Cpf1 endonuclease and corresponding guide RNAsand PAM sites are disclosed in US Patent Application Publication2016/0208243 A1. Other CRISPR nucleases useful for editing genomesinclude Cas12b and Cas12c (see Shmakov et al. (2015) Mol. Cell,60:385-397; Harrington et al. (2020) Molecular Celldoi:10.1016/j.molcel.2020.06.022) and CasX and CasY (see Burstein et al.(2016) Nature, doi:10.1038/nature21059; Harrington et al. (2020)Molecular Cell doi:10.1016/j.molcel.2020.06.022), or Cas12j (Pausch etal, (2020) Science 10.1126/science.abb1400). Plant RNA promoters forexpressing CRISPR guide RNA and plant codon-optimized CRISPR Cas9endonuclease are disclosed in International Patent ApplicationPCT/US2015/018104 (published as WO 2015/131101 and claiming priority toU.S. Provisional Patent Application 61/945,700). Methods of using CRISPRtechnology for genome editing in plants are disclosed in US PatentApplication Publications US 2015/0082478A1 and US 2015/0059010A1 and inInternational Patent Application PCT/US2015/038767 A1 (published as WO2016/007347 and claiming priority to U.S.

Provisional Patent Application 62/023,246). All of the patentpublications referenced in this paragraph are incorporated herein byreference in their entirety. In certain embodiments, an RNA-guidedendonuclease that leaves a blunt end following cleavage of the targetsite is used. Blunt-end cutting RNA-guided endonucleases include Cas9,Cas12c, and Cas 12h (Yan et al., 2019). In certain embodiments, anRNA-guided endonuclease that leaves a staggered single stranded DNAoverhanging end following cleavage of the target site following cleavageof the target site is used. Staggered-end cutting RNA-guidedendonucleases include Cas12a, Cas12b, and Cas12e.

The methods can also use sequence-specific endonucleases orsequence-specific endonucleases and guide RNAs that cleave a single DNAstrand in a dsDNA target site. Such cleavage of a single DNA strand in adsDNA target site is also referred to herein and elsewhere as “nicking”and can be effected by various “nickases” or systems that provide fornicking. Nickases that can be used include nCas9 (Cas9 comprising a D10Aamino acid substitution), nCas12a (e.g., Cas12a comprising an R1226Aamino acid substitution; Yamano et al., 2016), Cas12i (Yan et al. 2019),a zinc finger nickase e.g., as disclosed in Kim et al., 2012), a TALEnickase (e.g., as disclosed in Wu et al., 2014), or a combinationthereof. In certain embodiments, systems that provide for nicking cancomprise a Cas nuclease (e.g., Cas9 and/or Cas12a) and guide RNAmolecules that have at least one base mismatch to DNA sequences in thetarget editing site (Fu et al., 2019). In certain embodiments, genomemodifications can be introduced into the target editing site by creatingsingle stranded breaks (i.e., “nicks”) in genomic locations separated byno more than about 10, 20, 30, 40, 50, 60, 80, 100, 150, or 200 basepairs of DNA. In certain illustrative and non-limiting embodiments, twonickases (i.e., a CAS nuclease which introduces a single stranded DNAbreak including nCas9, nCas12a, Cas12i, zinc finger nickases, TALEnickases, combinations thereof, and the like) or nickase systems candirected to make cuts to nearby sites separated by no more than about10, 20, 30, 40, 50, 60, 80 or 100 base pairs of DNA. In instances wherean RNA guided nickase and an RNA guide are used, the RNA guides areadjacent to PAM sequences that are sufficiently close (i.e., separatedby no more than about 10, 20, 30, 40, 50, 60, 80, 100, 150, or 200 basepairs of DNA). For the purposes of gene editing, CRISPR arrays can bedesigned to contain one or multiple guide RNA sequences corresponding toa desired target DNA sequence; see, for example, Cong et al. (2013)Science, 339:819-823; Ran et al. (2013) Nature Protocols, 8:2281-2308.At least 16 or 17 nucleotides of gRNA sequence are required by Cas9 forDNA cleavage to occur; for Cpf1 at least 16 nucleotides of gRNA sequenceare needed to achieve detectable DNA cleavage and at least 18nucleotides of gRNA sequence were reported necessary for efficient DNAcleavage in vitro; see Zetsche et al. (2015) Cell, 163:759-771. Inpractice, guide RNA sequences are generally designed to have a length of17-24 nucleotides (frequently 19, 20, or 21 nucleotides) and exactcomplementarity (i.e., perfect base-pairing) to the targeted gene ornucleic acid sequence; guide RNAs having less than 100% complementarityto the target sequence can be used (e.g., a gRNA with a length of 20nucleotides and 1-4 mismatches to the target sequence) but can increasethe potential for off-target effects. The design of effective guide RNAsfor use in plant genome editing is disclosed in US Patent ApplicationPublication 2015/0082478 A1, the entire specification of which isincorporated herein by reference. More recently, efficient gene editinghas been achieved using a chimeric “single guide RNA” (“sgRNA”), anengineered (synthetic) single RNA molecule that mimics a naturallyoccurring crRNA-tracrRNA complex and contains both a tracrRNA (forbinding the nuclease) and at least one crRNA (to guide the nuclease tothe sequence targeted for editing); see, for example, Cong et al. (2013)Science, 339:819-823; Xing et al. (2014) BMC Plant Biol., 14:327-340.Chemically modified sgRNAs have been demonstrated to be effective ingenome editing; see, for example, Hendel et al. (2015) NatureBiotechnol., 985-991. The design of effective gRNAs for use in plantgenome editing is disclosed in US Patent Application Publication2015/0082478 A1, the entire specification of which is incorporatedherein by reference.

Genomic DNA may also be modified via base editing. Both adenine baseeditors (ABE) which convert A/T base pairs to G/C base pairs in genomicDNA as well as cytosine base pair editors (CBE) which effect C to Tsubstitutions can be used in certain embodiments of the methods providedherein. In certain embodiments, useful ABE and CBE can comprise genomesite specific DNA binding elements (e.g., RNA-dependent DNA bindingproteins including catalytically inactive Cas9 and Cas12 proteins orCas9 and Cas12 nickases) operably linked to adenine or cytidinedeaminases and used with guide RNAs which position the protein near thenucleotide targeted for substitution. Suitable ABE and CBE disclosed inthe literature (Kim, Nat Plants, 2018 March; 4(3):148-151) can beadapted for use in the methods set forth herein. In certain embodiments,a CBE can comprise a fusion between a catalytically inactive Cas9(dCas9) RNA dependent DNA binding protein fused to a cytidine deaminasewhich converts cytosine (C) to uridine (U) and selected guide RNAs,thereby effecting a C to T substitution; see Komor et al. (2016) Nature,533:420-424. In other embodiments, C to T substitutions are effectedwith Cas9 nickase [Cas9n(D10A)] fused to an improved cytidine deaminaseand optionally a bacteriophage Mu dsDNA (double-stranded DNA)end-binding protein Gam; see Komor et al., Sci Adv. 2017 August;3(8):eaao4774. In other embodiments, adenine base editors (ABEs)comprising an adenine deaminase fused to catalytically inactive Cas9(dCas9) or a Cas9 D10A nickase can be used to convert A/T base pairs toG/C base pairs in genomic DNA (Gaudelli et al., (2017) Nature551(7681):464-471.

In certain embodiments, zinc finger nucleases or zinc finger nickasescan also be used in the methods provided herein. Zinc-finger nucleasesare site-specific endonucleases comprising two protein domains: aDNA-binding domain, comprising a plurality of individual zinc fingerrepeats that each recognize between 9 and 18 base pairs, and aDNA-cleavage domain that comprises a nuclease domain (typically Fokl).The cleavage domain dimerizes in order to cleave DNA; therefore, a pairof ZFNs are required to target non-palindromic target polynucleotides.In certain embodiments, zinc finger nuclease and zinc finger nickasedesign methods which have been described (Urnov et al. (2010) NatureRev. Genet., 11:636-646; Mohanta et al. (2017) Genes vol. 8, 12: 399;Ramirez et al. Nucleic Acids Res. (2012); 40(12): 5560-5568; Liu et al.(2013) Nature Communications, 4: 2565) can be adapted for use in themethods set forth herein. The zinc finger binding domains of the zincfinger nuclease or nickase provide specificity and can be engineered tospecifically recognize any desired target DNA sequence. The zinc fingerDNA binding domains are derived from the DNA-binding domain of a largeclass of eukaryotic transcription factors called zinc finger proteins(ZFPs). The DNA-binding domain of ZFPs typically contains a tandem arrayof at least three zinc “fingers” each recognizing a specific triplet ofDNA. A number of strategies can be used to design the bindingspecificity of the zinc finger binding domain. One approach, termed“modular assembly”, relies on the functional autonomy of individual zincfingers with DNA. In this approach, a given sequence is targeted byidentifying zinc fingers for each component triplet in the sequence andlinking them into a multifinger peptide. Several alternative strategiesfor designing zinc finger DNA binding domains have also been developed.These methods are designed to accommodate the ability of zinc fingers tocontact neighboring fingers as well as nucleotide bases outside theirtarget triplet. Typically, the engineered zinc finger DNA binding domainhas a novel binding specificity, compared to a naturally-occurring zincfinger protein. Engineering methods include, for example, rationaldesign and various types of selection. Rational design includes, forexample, the use of databases of triplet (or quadruplet) nucleotidesequences and individual zinc finger amino acid sequences, in which eachtriplet or quadruplet nucleotide sequence is associated with one or moreamino acid sequences of zinc fingers which bind the particular tripletor quadruplet sequence. See, e.g., U.S. Pat. Nos. 6,453,242 and6,534,261, both incorporated herein by reference in their entirety.Exemplary selection methods (e.g., phage display and yeast two-hybridsystems) can be adapted for use in the methods described herein. Inaddition, enhancement of binding specificity for zinc finger bindingdomains has been described in U.S. Pat. No. 6,794,136, incorporatedherein by reference in its entirety. In addition, individual zinc fingerdomains may be linked together using any suitable linker sequences.Examples of linker sequences are publicly known, e.g., see U.S. Pat.Nos. 6,479,626; 6,903,185; and 7,153,949, incorporated herein byreference in their entirety. The nucleic acid cleavage domain isnon-specific and is typically a restriction endonuclease, such as Fokl.This endonuclease must dimerize to cleave DNA. Thus, cleavage by Fokl aspart of a ZFN requires two adjacent and independent binding events,which must occur in both the correct orientation and with appropriatespacing to permit dimer formation. The requirement for two DNA bindingevents enables more specific targeting of long and potentially uniquerecognition sites. Fokl variants with enhanced activities have beendescribed and can be adapted for use in the methods described herein;see, e.g., Guo et al. (2010) J. Mol. Biol., 400:96-107.

Transcription activator like effectors (TALEs) are proteins secreted bycertain Xanthomonas species to modulate gene expression in host plantsand to facilitate the colonization by and survival of the bacterium.TALEs act as transcription factors and modulate expression of resistancegenes in the plants. Recent studies of TALEs have revealed the codelinking the repetitive region of TALEs with their target DNA-bindingsites. TALEs comprise a highly conserved and repetitive regionconsisting of tandem repeats of mostly 33 or 34 amino acid segments. Therepeat monomers differ from each other mainly at amino acid positions 12and 13. A strong correlation between unique pairs of amino acids atpositions 12 and 13 and the corresponding nucleotide in the TALE-bindingsite has been found. The simple relationship between amino acid sequenceand DNA recognition of the TALE binding domain allows for the design ofDNA binding domains of any desired specificity. TALEs can be linked to anon-specific DNA cleavage domain to prepare genome editing proteins,referred to as TAL-effector nucleases or TALENs. As in the case of ZFNs,a restriction endonuclease, such as Fokl, can be conveniently used.Methods for use of TALENs in plants have been described and can beadapted for use in the methods described herein, see Mahfouz et al.(2011) Proc. Natl. Acad. Sci. USA, 108:2623-2628; Mahfouz (2011) GMCrops, 2:99-103; and Mohanta et al. (2017) Genes vol. 8, 12: 399). TALEnickases have also been described and can be adapted for use in methodsdescribed herein (Wu et al.; Biochem Biophys Res Commun. (2014);446(1):261-6; Luo et al; Scientific Reports 6, Article number: 20657(2016)).

Embodiments of the donor DNA template molecule having a sequence that isintegrated at the site of at least one double-strand break (DSB) in agenome include double-stranded DNA, a single-stranded DNA, asingle-stranded DNA/RNA hybrid, and a double-stranded DNA/RNA hybrid. Inembodiments, a donor DNA template molecule that is a double-stranded(e.g., a dsDNA or dsDNA/RNA hybrid) molecule is provided directly to theplant protoplast or plant cell in the form of a double-stranded DNA or adouble-stranded DNA/RNA hybrid, or as two single-stranded DNA (ssDNA)molecules that are capable of hybridizing to form dsDNA, or as asingle-stranded DNA molecule and a single-stranded RNA (ssRNA) moleculethat are capable of hybridizing to form a double-stranded DNA/RNAhybrid; that is to say, the double-stranded polynucleotide molecule isnot provided indirectly, for example, by expression in the cell of adsDNA encoded by a plasmid or other vector. In various non-limitingembodiments of the method, the donor DNA template molecule that isintegrated (or that has a sequence that is integrated) at the site of atleast one double-strand break (DSB) in a genome is double-stranded andblunt-ended; in other embodiments the donor DNA template molecule isdouble-stranded and has an overhang or “sticky end” consisting ofunpaired nucleotides (e.g., 1, 2, 3, 4, 5, or 6 unpaired nucleotides) atone terminus or both termini. In an embodiment, the DSB in the genomehas no unpaired nucleotides at the cleavage site, and the donor DNAtemplate molecule that is integrated (or that has a sequence that isintegrated) at the site of the DSB is a blunt-ended double-stranded DNAor blunt-ended double-stranded DNA/RNA hybrid molecule, or alternativelyis a single-stranded DNA or a single-stranded DNA/RNA hybrid molecule.In another embodiment, the DSB in the genome has one or more unpairednucleotides at one or both sides of the cleavage site, and the donor DNAtemplate molecule that is integrated (or that has a sequence that isintegrated) at the site of the DSB is a double-stranded DNA ordouble-stranded DNA/RNA hybrid molecule with an overhang or “sticky end”consisting of unpaired nucleotides at one or both termini, oralternatively is a single-stranded DNA or a single-stranded DNA/RNAhybrid molecule; in embodiments, the donor DNA template molecule DSB isa double-stranded DNA or double-stranded DNA/RNA hybrid molecule thatincludes an overhang at one or at both termini, wherein the overhangconsists of the same number of unpaired nucleotides as the number ofunpaired nucleotides created at the site of a DSB by a nuclease thatcuts in an off-set fashion (e.g., where a Cas12 nuclease effects anoff-set DSB with 5-nucleotide overhangs in the genomic sequence, thedonor DNA template molecule that is to be integrated (or that has asequence that is to be integrated) at the site of the DSB isdouble-stranded and has 5 unpaired nucleotides at one or both termini).In certain embodiments, one or both termini of the donor DNA templatemolecule contain no regions of sequence homology (identity orcomplementarity) to genomic regions flanking the DSB; that is to say,one or both termini of the donor DNA template molecule contain noregions of sequence that is sufficiently complementary to permithybridization to genomic regions immediately adjacent to the location ofthe DSB. In embodiments, the donor DNA template molecule contains nohomology to the locus of the DSB, that is to say, the donor DNA templatemolecule contains no nucleotide sequence that is sufficientlycomplementary to permit hybridization to genomic regions immediatelyadjacent to the location of the DSB. In embodiments, the donor DNAtemplate molecule is at least partially double-stranded and includes2-20 base-pairs, e. g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 base-pairs; in embodiments, the donor DNA templatemolecule is double-stranded and blunt-ended and consists of 2-20base-pairs, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20 base-pairs; in other embodiments, the donor DNAtemplate molecule is double-stranded and includes 2-20 base-pairs, e.g.,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20base-pairs and in addition has at least one overhang or “sticky end”consisting of at least one additional, unpaired nucleotide at one or atboth termini. In an embodiment, the donor DNA template molecule that isintegrated (or that has a sequence that is integrated) at the site of atleast one double-strand break (DSB) in a genome is a blunt-endeddouble-stranded DNA or a blunt-ended double-stranded DNA/RNA hybridmolecule of about 18 to about 300 base-pairs, or about 20 to about 200base-pairs, or about 30 to about 100 base-pairs, and having at least onephosphorothioate bond between adjacent nucleotides at a 5′ end, 3′ end,or both 5′ and 3′ ends. In embodiments, the donor DNA template moleculeincludes single strands of at least 11, at least 18, at least 20, atleast 30, at least 40, at least 60, at least 80, at least 100, at least120, at least 140, at least 160, at least 180, at least 200, at least240, at about 280, or at least 320 nucleotides. In embodiments, thedonor DNA template molecule has a length of at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, atleast 10, or at least 11 base-pairs if double-stranded (or nucleotidesif single-stranded), or between about 2 to about 320 base-pairs ifdouble-stranded (or nucleotides if single-stranded), or between about 2to about 500 base-pairs if double-stranded (or nucleotides ifsingle-stranded), or between about 5 to about 500 base-pairs ifdouble-stranded (or nucleotides if single-stranded), or between about 5to about 300 base-pairs if double-stranded (or nucleotides ifsingle-stranded), or between about 11 to about 300 base-pairs ifdouble-stranded (or nucleotides if single-stranded), or about 18 toabout 300 base-pairs if double-stranded (or nucleotides ifsingle-stranded), or between about 30 to about 100 base-pairs ifdouble-stranded (or nucleotides if single-stranded). In embodiments, thedonor DNA template molecule includes chemically modified nucleotides(see, e.g., the various modifications of internucleotide linkages,bases, and sugars described in Verma and Eckstein (1998) Annu. Rev.Biochem., 67:99-134); in embodiments, the naturally occurringphosphodiester backbone of the donor DNA template molecule is partiallyor completely modified with phosphorothioate, phosphorodithioate, ormethylphosphonate internucleotide linkage modifications, or the donorDNA template molecule includes modified nucleoside bases or modifiedsugars, or the donor DNA template molecule is labelled with afluorescent moiety (e.g., fluorescein or rhodamine or a fluorescentnucleoside analogue) or other detectable label (e.g., biotin or anisotope). In another embodiment, the donor DNA template moleculecontains secondary structure that provides stability or acts as anaptamer. Other related embodiments include double-stranded DNA/RNAhybrid molecules, single-stranded DNA/RNA hybrid donor molecules, andsingle-stranded donor DNA template molecules (including single-stranded,chemically modified donor DNA template molecules), which in analogousprocedures are integrated (or have a sequence that is integrated) at thesite of a double-strand break. Donor DNA templates provided hereininclude those comprising CgRRS sequences flanked by DNA with homology toa donor DNA template (e.g., SEQ ID NO: 32). In certain embodiments,integration of the donor DNA templates can be facilitated by use of abacteriophage lambda exonuclease, a bacteriophage lambda beta SSAPprotein, and an E. coli SSB essentially as set forth in US PatentApplication Publication 20200407754, which is incorporated herein byreference in its entirety.

Donor DNA template molecules used in the methods provided herein includeDNA molecules comprising, from 5′ to 3′, a first homology arm, areplacement DNA, and a second homology arm, wherein the homology armscontaining sequences that are partially or completely homologous togenomic DNA (gDNA) sequences flanking a target site-specificendonuclease cleavage site in the gDNA. In certain embodiments, thereplacement DNA can comprise an insertion, deletion, or substitution of1 or more DNA base pairs relative to the target gDNA. In an embodiment,the donor DNA template molecule is double-stranded and perfectlybase-paired through all or most of its length, with the possibleexception of any unpaired nucleotides at either terminus or bothtermini. In another embodiment, the donor DNA template molecule isdouble-stranded and includes one or more non-terminal mismatches ornon-terminal unpaired nucleotides within the otherwise double-strandedduplex. In an embodiment, the donor DNA template molecule that isintegrated at the site of at least one double-strand break (DSB)includes between 2-20 nucleotides in one (if single-stranded) or in bothstrands (if double-stranded), e. g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 nucleotides on one or on both strands,each of which can be base-paired to a nucleotide on the opposite strand(in the case of a perfectly base-paired double-stranded polynucleotidemolecule). Such donor DNA templates can be integrated in genomic DNAcontaining blunt and/or staggered double stranded DNA breaks byhomology-directed repair (HDR). In certain embodiments, a donor DNAtemplate homology arm can be about 20, 50, 100, 200, 400, or 600 toabout 800, or 1000 base pairs in length. In certain embodiments, a donorDNA template molecule can be delivered to a plant cell) in a circular(e.g., a plasmid or a viral vector including a geminivirus vector) or alinear DNA molecule. In certain embodiments, a circular or linear DNAmolecule that is used can comprise a modified donor DNA templatemolecule comprising, from 5′ to 3′, a first copy of the targetsequence-specific endonuclease cleavage site sequence, the firsthomology arm, the replacement DNA, the second homology arm, and a secondcopy of the target sequence-specific endonuclease cleavage sitesequence. Without seeking to be limited by theory, such modified donorDNA template molecules can be cleaved by the same sequence-specificendonuclease that is used to cleave the target site gDNA of theeukaryotic cell to release a donor DNA template molecule that canparticipate in HDR-mediated genome modification of the target editingsite in the plant cell genome. In certain embodiments, the donor DNAtemplate can comprise a linear DNA molecule comprising, from 5′ to 3′, acleaved target sequence-specific endonuclease cleavage site sequence,the first homology arm, the replacement DNA, the second homology arm,and a cleaved target sequence-specific endonuclease cleavage sitesequence. In certain embodiments, the cleaved target sequence-specificendonuclease sequence can comprise a blunt DNA end or a blunt DNA endthat can optionally comprise a 5′ phosphate group. In certainembodiments, the cleaved target sequence-specific endonuclease sequencecomprises a DNA end having a single-stranded 5′ or 3′ DNA overhang. Suchcleaved target sequence-specific endonuclease cleavage site sequencescan be produced by either cleaving an intact target sequence-specificendonuclease cleavage site sequence or by synthesizing a copy of thecleaved target sequence-specific endonuclease cleavage site sequence.Donor DNA templates can be synthesized either chemically orenzymatically (e.g., in a polymerase chain reaction (PCR)). Donor DNAtemplates provided herein include those comprising CgRRS sequencesflanked by DNA with homology to a donor DNA template e (e.g., SEQ ID NO:32).

Various treatments are useful in delivery of gene editing moleculesand/or other molecules to a MIR162 or INIR12 plant cell. In certainembodiments, one or more treatments is employed to deliver the geneediting or other molecules (e.g., comprising a polynucleotide,polypeptide or combination thereof) into a eukaryotic or plant cell,e.g., through barriers such as a cell wall, a plasma membrane, a nuclearenvelope, and/or other lipid bilayer. In certain embodiments, apolynucleotide-, polypeptide-, or RNP-containing composition comprisingthe molecules are delivered directly, for example by direct contact ofthe composition with a plant cell. Aforementioned compositions can beprovided in the form of a liquid, a solution, a suspension, an emulsion,a reverse emulsion, a colloid, a dispersion, a gel, liposomes, micelles,an injectable material, an aerosol, a solid, a powder, a particulate, ananoparticle, or a combination thereof can be applied directly to aplant, plant part, plant cell, or plant explant (e.g., through abrasionor puncture or otherwise disruption of the cell wall or cell membrane,by spraying or dipping or soaking or otherwise directly contacting, bymicroinjection). For example, a plant cell or plant protoplast is soakedin a liquid genome editing molecule-containing composition, whereby theagent is delivered to the plant cell. In certain embodiments, theagent-containing composition is delivered using negative or positivepressure, for example, using vacuum infiltration or application ofhydrodynamic or fluid pressure. In certain embodiments, theagent-containing composition is introduced into a plant cell or plantprotoplast, e.g., by microinjection or by disruption or deformation ofthe cell wall or cell membrane, for example by physical treatments suchas by application of negative or positive pressure, shear forces, ortreatment with a chemical or physical delivery agent such assurfactants, liposomes, or nanoparticles; see, e.g., delivery ofmaterials to cells employing microfluidic flow through a cell-deformingconstriction as described in US Published Patent Application2014/0287509, incorporated by reference in its entirety herein. Othertechniques useful for delivering the agent-containing composition to aeukaryotic cell, plant cell or plant protoplast include: ultrasound orsonication; vibration, friction, shear stress, vortexing, cavitation;centrifugation or application of mechanical force; mechanical cell wallor cell membrane deformation or breakage; enzymatic cell wall or cellmembrane breakage or permeabilization; abrasion or mechanicalscarification (e.g., abrasion with carborundum or other particulateabrasive or scarification with a file or sandpaper) or chemicalscarification (e.g., treatment with an acid or caustic agent); andelectroporation. In certain embodiments, the agent-containingcomposition is provided by bacterially mediated (e.g., Agrobacteriumsp., Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobiumsp., Azobacter sp., Phyllobacterium sp.) transfection of the plant cellor plant protoplast with a polynucleotide encoding the genome editingmolecules (e.g., RNA dependent DNA endonuclease, RNA dependent DNAbinding protein, RNA dependent nickase, ABE, or CBE, and/or guide RNA);see, e.g., Broothaerts et al. (2005) Nature, 433:629-633). Any of thesetechniques or a combination thereof are alternatively employed on theplant explant, plant part or tissue or intact plant (or seed) from whicha plant cell is optionally subsequently obtained or isolated; in certainembodiments, the agent-containing composition is delivered in a separatestep after the plant cell has been isolated.

In some embodiments, one or more polynucleotides or vectors drivingexpression of one or more genome editing molecules or trait-conferringgenes (e.g.; herbicide tolerance, insect resistance, and/or malesterility) are introduced into a MIR162 or INIR12 plant cell. In certainembodiments, a polynucleotide vector comprises a regulatory element suchas a promoter operably linked to one or more polynucleotides encodinggenome editing molecules and/or trait-conferring genes. In suchembodiments, expression of these polynucleotides can be controlled byselection of the appropriate promoter, particularly promoters functionalin a eukaryotic cell (e.g., plant cell); useful promoters includeconstitutive, conditional, inducible, and temporally or spatiallyspecific promoters (e.g., a tissue specific promoter, a developmentallyregulated promoter, or a cell cycle regulated promoter). Developmentallyregulated promoters that can be used in plant cells include PhospholipidTransfer Protein (PLTP), fructose-1,6-bisphosphatase protein,NAD(P)-binding Rossmann-Fold protein, adipocyte plasmamembrane-associated protein-like protein, Rieske [2Fe-2S] iron-sulfurdomain protein, chlororespiratory reduction 6 protein, D-glycerate3-kinase, chloroplastic-like protein, chlorophyll a-b binding protein 7,chloroplastic-like protein, ultraviolet-B-repressible protein, Soulheme-binding family protein, Photosystem I reaction center subunit psi-Nprotein, and short-chain dehydrogenase/reductase protein that aredisclosed in US Patent Application Publication No. 20170121722, which isincorporated herein by reference in its entirety and specifically withrespect to such disclosure. In certain embodiments, the promoter isoperably linked to nucleotide sequences encoding multiple guide RNAs,wherein the sequences encoding guide RNAs are separated by a cleavagesite such as a nucleotide sequence encoding a microRNArecognition/cleavage site or a self-cleaving ribozyme (see, e.g.,Ferré-D'Amaré and Scott (2014) Cold Spring Harbor Perspectives Biol.,2:a003574). In certain embodiments, the promoter is an RNA polymeraseIII promoter operably linked to a nucleotide sequence encoding one ormore guide RNAs. In certain embodiments, the RNA polymerase III promoteris a plant U6 spliceosomal RNA promoter, which can be native to thegenome of the plant cell or from a different species, e.g., a U6promoter from maize, tomato, or soybean such as those disclosed U.S.Patent Application Publication 2017/0166912, or a homologue thereof; inan example, such a promoter is operably linked to DNA sequence encodinga first RNA molecule including a Cas12a gRNA followed by an operablylinked and suitable 3′ element such as a U6 poly-T terminator. Inanother embodiment, the RNA polymerase III promoter is a plant U3, 7SL(signal recognition particle RNA), U2, or U5 promoter, or chimericsthereof, e.g., as described in U.S. Patent Application Publication20170166912. In certain embodiments, the promoter operably linked to oneor more polynucleotides is a constitutive promoter that drives geneexpression in eukaryotic cells (e.g., plant cells). In certainembodiments, the promoter drives gene expression in the nucleus or in anorganelle such as a chloroplast or mitochondrion. Examples ofconstitutive promoters for use in plants include a CaMV 35S promoter asdisclosed in U.S. Pat. Nos. 5,858,742 and 5,322,938, a rice actinpromoter as disclosed in U.S. Pat. No. 5,641,876, a maize chloroplastaldolase promoter as disclosed in U.S. Pat. No. 7,151,204, and thenopaline synthase (NOS) and octopine synthase (OCS) promoters fromAgrobacterium tumefaciens. In certain embodiments, the promoter operablylinked to one or more polynucleotides encoding elements of agenome-editing system is a promoter from figwort mosaic virus (FMV), aRUBISCO promoter, or a pyruvate phosphate dikinase (PPDK) promoter,which is active in photosynthetic tissues. Other contemplated promotersinclude cell-specific or tissue-specific or developmentally regulatedpromoters, for example, a promoter that limits the expression of thenucleic acid targeting system to germline or reproductive cells (e.g.,promoters of genes encoding DNA ligases, recombinases, replicases, orother genes specifically expressed in germline or reproductive cells).In certain embodiments, the genome alteration is limited only to thosecells from which DNA is inherited in subsequent generations, which isadvantageous where it is desirable that expression of the genome-editingsystem be limited in order to avoid genotoxicity or other unwantedeffects. All of the patent publications referenced in this paragraph areincorporated herein by reference in their entirety.

Expression vectors or polynucleotides provided herein may contain a DNAsegment near the 3′ end of an expression cassette that acts as a signalto terminate transcription and directs polyadenylation of the resultantmRNA and may also support promoter activity. Such a 3′ element iscommonly referred to as a “3′-untranslated region” or “3′-UTR” or a“polyadenylation signal.” In some cases, plant gene-based 3′ elements(or terminators) consist of both the 3′-UTR and downstreamnon-transcribed sequence (Nuccio et al., 2015). Useful 3′ elementsinclude: Agrobacterium tumefaciens nos 3′, tml 3′, tmr 3′, tms 3′, ocs3′, and tr7 3′ elements disclosed in U.S. Pat. No. 6,090,627,incorporated herein by reference, and 3′ elements from plant genes suchas the heat shock protein 17, ubiquitin, and fructose-1,6-biphosphatasegenes from wheat (Triticum aestivum), and the glutelin, lactatedehydrogenase, and beta-tubulin genes from rice (Oryza sativa),disclosed in US Patent Application Publication 2002/0192813 A1. All ofthe patent publications referenced in this paragraph are incorporatedherein by reference in their entireties.

In certain embodiments, the MIR162 or INIR12 plant cells used herein cancomprise haploid, diploid, or polyploid plant cells or plantprotoplasts, for example, those obtained from a haploid, diploid, orpolyploid plant, plant part or tissue, or callus. In certainembodiments, plant cells in culture (or the regenerated plant, progenyseed, and progeny plant) are haploid or can be induced to becomehaploid; techniques for making and using haploid plants and plant cellsare known in the art, see, e.g., methods for generating haploids inArabidopsis thaliana by crossing of a wild-type strain to ahaploid-inducing strain that expresses altered forms of thecentromere-specific histone CENH3, as described by Maruthachalam andChan in “How to make haploid Arabidopsis thaliana”, protocol availableatwww[dot]openwetware[dot]org/images/d/d3/Haploid_Arabidopsis_protocol[dot]pdf;(Ravi et al. (2014) Nature Communications, 5:5334, doi:10.1038/ncomms6334). Haploids can also be obtained in a wide variety ofmonocot plants (e.g., maize, wheat, rice, sorghum, barley) by crossing aplant comprising a mutated CENH3 gene with a wildtype diploid plant togenerate haploid progeny as disclosed in U.S. Pat. No. 9,215,849, whichis incorporated herein by reference in its entirety. Haploid-inducingmaize lines that can be used to obtain haploid maize plants and/or cellsinclude Stock 6, MHI (Moldovian Haploid Inducer), indeterminategametophyte (ig) mutation, KEMS, RWK, ZEM, ZMS, KMS, and well astransgenic haploid inducer lines disclosed in U.S. Pat. No. 9,677,082,which is incorporated herein by reference in its entirety. Examples ofhaploid cells include but are not limited to plant cells obtained fromhaploid plants and plant cells obtained from reproductive tissues, e.g.,from flowers, developing flowers or flower buds, ovaries, ovules,megaspores, anthers, pollen, megagametophyte, and microspores. Incertain embodiments where the plant cell or plant protoplast is haploid,the genetic complement can be doubled by chromosome doubling (e.g., byspontaneous chromosomal doubling by meiotic non-reduction, or by using achromosome doubling agent such as colchicine, oryzalin, trifluralin,pronamide, nitrous oxide gas, anti-microtubule herbicides,anti-microtubule agents, and mitotic inhibitors) in the plant cell orplant protoplast to produce a doubled haploid plant cell or plantprotoplast wherein the complement of genes or alleles is homozygous; yetother embodiments include regeneration of a doubled haploid plant fromthe doubled haploid plant cell or plant protoplast. Another embodimentis related to a hybrid plant having at least one parent plant that is adoubled haploid plant provided by this approach. Production of doubledhaploid plants provides homozygosity in one generation, instead ofrequiring several generations of self-crossing to obtain homozygousplants. The use of doubled haploids is advantageous in any situationwhere there is a desire to establish genetic purity (i.e., homozygosity)in the least possible time. Doubled haploid production can beparticularly advantageous in slow-growing plants or for producing hybridplants that are offspring of at least one doubled-haploid plant.

In certain embodiments, the MIR162 or INIR12 plant cells used in themethods provided herein can include non-dividing cells. Suchnon-dividing cells can include plant cell protoplasts, plant cellssubjected to one or more of a genetic and/or pharmaceutically-inducedcell-cycle blockage, and the like.

In certain embodiments, the MIR162 or INIR12 plant cells in used in themethods provided herein can include dividing cells. Dividing cells caninclude those cells found in various plant tissues including leaves,meristems, and embryos. These tissues include but are not limited todividing cells from young maize leaf, meristems and scutellar tissuefrom about 8 or 10 to about 12 or 14 days after pollination (DAP)embryos. The isolation of maize embryos has been described in severalpublications (Brettschneider, Becker, and Lörz 1997; Leduc et al. 1996;Frame et al. 2011; K. Wang and Frame 2009). In certain embodiments,basal leaf tissues (e.g., leaf tissues located about 0 to 3 cm from theligule of a maize plant; Kirienko, Luo, and Sylvester 2012) are targetedfor HDR-mediated gene editing. Methods for obtaining regenerable plantstructures and regenerating plants from the NHEJ-, MMEJ-, orHDR-mediated gene editing of plant cells provided herein can be adaptedfrom methods disclosed in US Patent Application Publication No.20170121722, which is incorporated herein by reference in its entiretyand specifically with respect to such disclosure. In certainembodiments, single plant cells subjected to the HDR-mediated geneediting will give rise to single regenerable plant structures. Incertain embodiments, the single regenerable plant cell structure canform from a single cell on, or within, an explant that has beensubjected to the NHEJ-, MMEJ-, or HDR-mediated gene editing.

In some embodiments, methods provided herein can include the additionalstep of growing or regenerating an INIR12 plant from a INIR12 plant cellthat had been subjected to the gene editing or from a regenerable plantstructure obtained from that INIR12 plant cell. In certain embodiments,the plant can further comprise an inserted transgene, a target geneedit, or genome edit as provided by the methods and compositionsdisclosed herein. In certain embodiments, callus is produced from theplant cell, and plantlets and plants produced from such callus. In otherembodiments, whole seedlings or plants are grown directly from the plantcell without a callus stage. Thus, additional related aspects aredirected to whole seedlings and plants grown or regenerated from theplant cell or plant protoplast having a target gene edit or genome edit,as well as the seeds of such plants. In certain embodiments wherein theplant cell or plant protoplast is subjected to genetic modification (forexample, genome editing by means of, e.g., an RdDe), the grown orregenerated plant exhibits a phenotype associated with the geneticmodification. In certain embodiments, the grown or regenerated plantincludes in its genome two or more genetic or epigenetic modificationsthat in combination provide at least one phenotype of interest. Incertain embodiments, a heterogeneous population of plant cells having atarget gene edit or genome edit, at least some of which include at leastone genetic or epigenetic modification, is provided by the method;related aspects include a plant having a phenotype of interestassociated with the genetic or epigenetic modification, provided byeither regeneration of a plant having the phenotype of interest from aplant cell or plant protoplast selected from the heterogeneouspopulation of plant cells having a target gene or genome edit, or byselection of a plant having the phenotype of interest from aheterogeneous population of plants grown or regenerated from thepopulation of plant cells having a targeted genetic edit or genome edit.Examples of phenotypes of interest include herbicide resistance,improved tolerance of abiotic stress (e.g., tolerance of temperatureextremes, drought, or salt) or biotic stress (e.g., resistance tonematode, bacterial, or fungal pathogens), improved utilization ofnutrients or water, modified lipid, carbohydrate, or proteincomposition, improved flavor or appearance, improved storagecharacteristics (e.g., resistance to bruising, browning, or softening),increased yield, altered morphology (e.g., floral architecture or color,plant height, branching, root structure). In an embodiment, aheterogeneous population of plant cells having a target gene edit orgenome edit (or seedlings or plants grown or regenerated therefrom) isexposed to conditions permitting expression of the phenotype ofinterest; e.g., selection for herbicide resistance can include exposingthe population of plant cells having a target gene edit or genome edit(or seedlings or plants grown or regenerated therefrom) to an amount ofherbicide or other substance that inhibits growth or is toxic, allowingidentification and selection of those resistant plant cells (orseedlings or plants) that survive treatment. Methods for obtainingregenerable plant structures and regenerating plants from plant cells orregenerable plant structures can be adapted from published procedures(Roest and Gilissen, Acta Bot. Neerl., 1989, 38(1), 1-23; Bhaskaran andSmith, Crop Sci. 30(6):1328-1337; Ikeuchi et al., Development, 2016,143: 1442-1451). Methods for obtaining regenerable plant structures andregenerating plants from plant cells or regenerable plant structures canalso be adapted from US Patent Application Publication No. 20170121722,which is incorporated herein by reference in its entirety andspecifically with respect to such disclosure. Also provided areheterogeneous or homogeneous populations of such plants or parts thereof(e.g., seeds), succeeding generations or seeds of such plants grown orregenerated from the plant cells or plant protoplasts, having a targetgene edit or genome edit. Additional related aspects include a hybridplant provided by crossing a first plant grown or regenerated from aplant cell or plant protoplast having a target gene edit or genome editand having at least one genetic or epigenetic modification, with asecond plant, wherein the hybrid plant contains the genetic orepigenetic modification; also contemplated is seed produced by thehybrid plant. Also envisioned as related aspects are progeny seed andprogeny plants, including hybrid seed and hybrid plants, having theregenerated plant as a parent or ancestor. The plant cells andderivative plants and seeds disclosed herein can be used for variouspurposes useful to the consumer or grower. In other embodiments,processed products are made from the INIR12 plant or its seeds,including: (a) maize seed meal (defatted or non-defatted); (b) extractedproteins, oils, sugars, and starches; (c) fermentation products; (d)animal feed or human food products (e.g., feed and food comprising maizeseed meal (defatted or non-defatted) and other ingredients (e.g., othercereal grains, other seed meal, other protein meal, other oil, otherstarch, other sugar, a binder, a preservative, a humectant, a vitamin,and/or mineral; (e) a pharmaceutical; (f) raw or processed biomass(e.g., cellulosic and/or lignocellulosic material); and (g) variousindustrial products.

Embodiments

Various embodiments of the plants, genomes, methods, biological samples,and other compositions described herein are set forth in the followingsets of numbered embodiments.

1a. A transgenic maize plant cell comprising a first ZmUbiInt promoter,a vip3Aa19 or vip3Aa20 coding region which is operably linked to saidpromoter, a CaMV 35S terminator element which is operably linked to saidvip3Aa19 or vip3Aa20 coding region, and a nopaline synthase terminatorelement, wherein said cell does not contain a second ZmUbiInt promoterand an operably linked phosphomannose isomerase coding region betweensaid terminator elements, optionally wherein: (i) the ZmUbiInt promoter,the vip3Aa19 or vip3Aa20 coding region which is operably linked to saidpromoter, the CaMV 35S terminator element which is operably linked tosaid vip3Aa19 or vip3Aa20 coding region are located in the maize plantcell genomic location of the MIR162 transgenic locus; (ii) wherein aselectable marker or scoreable is absent from said maize plant cellgenomic location, and/or (iii) wherein the nopaline synthase terminatorelement is not separated from the CaMV 35S terminator element by DNAencoding a selectable marker protein, a scoreable marker protein, or aprotein conferring a useful trait.

1b. A transgenic maize plant cell comprising a nucleotide sequencecomprising a first ZmUbiInt promoter, a vip3Aa19 or vip3Aa20 codingregion which is operably linked to said promoter, a CaMV 35S terminatorelement which is operably linked to said vip3Aa19 or vip3Aa20 codingregion, and a nopaline synthase terminator element, wherein saidnucleotide sequence does not contain a second ZmUbiInt promoter and anoperably linked phosphomannose isomerase coding region between saidterminator elements optionally wherein: (i) the ZmUbiInt promoter, thevip3Aa19 or vip3Aa20 coding region which is operably linked to saidpromoter, the CaMV 35S terminator element which is operably linked tosaid vip3Aa19 or vip3Aa20 coding region are located in the maize plantcell genomic location of the MIR162 transgenic locus; (ii) wherein aselectable marker or scoreable is absent from said maize plant cellgenomic location, and/or (iii) wherein the nopaline synthase terminatorelement is not separated from the CaMV 35S terminator element by DNAencoding a selectable marker protein, a scoreable marker protein, or aprotein conferring a useful trait.

1c. A transgenic maize plant cell comprising a nucleotide sequencecomprising a ZmUbiInt promoter, a vip3Aa19 or vip3Aa20 coding regionwhich is operably linked to said promoter, a CaMV 35S terminator elementwhich is operably linked to said vip3Aa19 or vip3Aa20 coding region, anda nopaline synthase terminator element, wherein said nucleotide sequencedoes not contain a phosphomannose isomerase coding region between saidterminator elements, optionally wherein: (i) the ZmUbiInt promoter, thevip3Aa19 or vip3Aa20 coding region which is operably linked to saidpromoter, the CaMV 35S terminator element which is operably linked tosaid vip3Aa19 or vip3Aa20 coding region are located in the maize plantcell genomic location of the MIR162 transgenic locus; (ii) wherein aselectable marker or scoreable is absent from said maize plant cellgenomic location, and/or (iii) wherein the nopaline synthase terminatorelement is not separated from the CaMV 35S terminator element by DNAencoding a selectable marker protein, a scoreable marker protein, or aprotein conferring a useful trait.

1d. A transgenic maize plant cell comprising a nucleotide sequencecomprising a first ZmUbiInt promoter, a vip3Aa19 or vip3Aa20 codingregion which is operably linked to said promoter, a CaMV 35S terminatorelement which is operably linked to said vip3Aa19 or vip3Aa20 codingregion, and a nopaline synthase terminator element, wherein saidnucleotide sequence does not contain a second ZmUbiInt promoter and anoperably linked phosphomannose isomerase coding region, optionallywherein: (i) the ZmUbiInt promoter, the vip3Aa19 or vip3Aa20 codingregion which is operably linked to said promoter, the CaMV 35Sterminator element which is operably linked to said vip3Aa19 or vip3Aa20coding region are located in the maize plant cell genomic location ofthe MIR162 transgenic locus; (ii) wherein a selectable marker orscoreable is absent from said maize plant cell genomic location, and/or(iii) wherein the nopaline synthase terminator element is not separatedfrom the CaMV 35S terminator element by DNA encoding a selectable markerprotein, a scoreable marker protein, or a protein conferring a usefultrait.

1e. A transgenic maize plant cell comprising a nucleotide sequencecomprising a ZmUbiInt promoter, a vip3Aa19 or vip3Aa20 coding regionwhich is operably linked to said promoter, a CaMV 35S terminator elementwhich is operably linked to said vip3Aa19 or vip3Aa20 coding region, anda nopaline synthase terminator element, wherein said nucleotide sequencedoes not contain a phosphomannose isomerase coding region, optionallywherein: (i) the ZmUbiInt promoter, the vip3Aa19 or vip3Aa20 codingregion which is operably linked to said promoter, the CaMV 35Sterminator element which is operably linked to said vip3Aa19 or vip3Aa20coding region are located in the maize plant cell genomic location ofthe MIR162 transgenic locus; (ii) wherein a selectable marker orscoreable is absent from said maize plant cell genomic location, and/or(iii) wherein the nopaline synthase terminator element is not separatedfrom the CaMV 35S terminator element by DNA encoding a selectable markerprotein, a scoreable marker protein, or a protein conferring a usefultrait.

1f. A transgenic maize plant cell comprising an INIR12 transgenic locuscomprising the first ZmUbiInt promoter, the vip3Aa19 or vip3Aa20 codingregion which is operably linked to said promoter, the CaMV 35Sterminator element which is operably linked to said vip3Aa19 or vip3Aa20coding region, and the nopaline synthase terminator element of a MIR162transgenic locus, allelic variants thereof, or other variants thereof,wherein DNA of said original MIR162 transgenic locus, allelic variantsthereof, or other variants thereof comprising a second ZmUbiInt promoterand an operably linked phosphomannose isomerase coding region is absent.

1g. A transgenic maize plant cell comprising an INIR12 transgenic locuscomprising an insertion and/or substitution of a DNA element comprisinga cognate guide RNA recognition site (CgRRS) in a DNA junctionpolynucleotide of said INIR12 transgenic locus.

2. The transgenic maize plant cell of embodiment 1a, 1b, 1c, 1d, 1e, or1f, wherein said INIR12 transgenic locus comprises DNA corresponding toat least nucleotide number 1101 to 5830 of SEQ ID NO:1 or SEQ ID NO:46and nucleotide number 9111 to 9360 of SEQ ID NO:1 or SEQ ID NO:46,wherein nucleotides corresponding to at least 5850 to 9090 of SEQ IDNO:1 or SEQ ID NO:46 are absent.

3. The transgenic maize plant cell of embodiment 1a, 1b, 1c, 1d, 1e, 1f,or 1g, wherein said INIR12 transgenic locus comprises the DNA moleculeset forth in SEQ ID NO: 2, 6, 29, 43, 44, 45, or 47.

4. The transgenic maize plant cell of embodiment 1a, 1b, 1c, 1d, 1e, or1f, wherein said INIR12 transgenic locus comprises:

(a) the DNA molecule set forth in SEQ ID NO: 3 wherein nucleotideresidues 1081 to 1104 are: (i) each either absent or independentlyselected from a guanine, a cytosine, an adenine residue, or a thymineresidue, with the proviso that nucleotides corresponding to residues1081 to 1104 of SEQ ID NO: 3 are not identical to residues 1081 to 1104of SEQ ID NO:1 or SEQ ID NO:46; (ii) comprise about 2 to 8 consecutiveresidues of nucleotides 1081 to 1092 of SEQ ID NO:1 or SEQ ID NO:46and/or about 2 to 8 consecutive residues of nucleotides 1093 to 1104 ofSEQ ID NO:1 or SEQ ID NO:46, with the proviso that nucleotidescorresponding to residues 1081 to 1104 of SEQ ID NO: 3 are not identicalto residues 1081 to 1104 of SEQ ID NO:1 or SEQ ID NO:46; (iii) anycombination of (i) and (ii); (v) are set forth in SEQ ID NO: 7, whereinn is absent, is independently selected from A, C, G, or T, correspond to1 to 10 residues of nucleotides 1083 to 1092 of SEQ ID NO:1 or SEQ IDNO:46, and/or correspond to 1 to 10 residues of nucleotides 1093 to 1102of SEQ ID NO:1 or SEQ ID NO:46 with the proviso that nucleotidescorresponding to nucleotide 3 to 22 of SEQ ID NO: 7 are not identical toresidues 1083 to 1102 of SEQ ID NO:1 or SEQ ID NO:46; (vi) are set forthin SEQ ID NO: 8; wherein n is absent, is independently selected from A,C, G, or T, correspond to 1 to 5 residues of nucleotides 1088 to 1092 ofSEQ ID NO:1 or SEQ ID NO:46, and/or correspond to 1 to 5 residues ofnucleotides 1093 to 1097 of SEQ ID NO:1 or SEQ ID NO:46 with the provisothat nucleotides corresponding to residues 8 to 17 of SEQ ID NO: 8 arenot identical to residues 1088 to 1097 of SEQ ID NO:1 or SEQ ID NO:46;(vii) are set forth in SEQ ID NO: 9; wherein n is absent, isindependently selected from A, C, G, or T, correspond to 1 to 3 residuesof nucleotides 1090-1092 of SEQ ID NO:1 or SEQ ID NO:46, and/orcorrespond to 1 to 3 residues of nucleotides 1093 to 1095 of SEQ ID NO:1or SEQ ID NO:46 with the proviso that nucleotides corresponding toresidues 1090 to 1095 of SEQ ID NO: 9 are not identical to nucleotides1090 to 1095 of SEQ ID NO:1 or SEQ ID NO:46); or (viii) are set forth inSEQ ID NO:1 or SEQ ID NO:460, 11, 12, 13, 14, 15, 16, 17, 18, or 19 andwherein nucleotides 5831 to 5842 of SEQ ID NO: 3 are each either absent,independently selected from a guanine, a cytosine, an adenine residue,or a thymine residue, comprise or consist of 1 or more nucleotidescorresponding to nucleotides 5831 to 5836 of SEQ ID NO:1 or SEQ IDNO:46, and/or comprise or consist of 1 or more nucleotides correspondingto nucleotides 9102 to 9107 of SEQ ID NO:1 or SEQ ID NO:46.

5. The transgenic maize plant cell of embodiment 1a, 1b, 1c, 1d, 1e, or1f, wherein said INIR12 transgenic locus further comprises an insertionand/or substitution of a DNA element comprising a cognate guide RNArecognition site (CgRRS) in a DNA junction polynucleotide of said INIR12transgenic locus.

6. The transgenic maize plant cell of embodiment 1g or 5, wherein saidcognate guide RNA recognition site (CgRRS) comprises SEQ ID NO: 26, 27,or 28, wherein the insertion and/or substitution is in a 5′ junctionpolynucleotide of said INIR12 transgenic locus and optionally whereinthe insertion and/or substitution is in a 5′ junction polynucleotide ofthe INIR12 transgenic locus corresponding to at least one of nucleotides1079 to 1098 of SEQ ID NO:1 or SEQ ID NO:46.

7. The transgenic maize plant cell of embodiment 6, wherein said CgRRScomprises the DNA molecule set forth in SEQ ID NO: 37.

8. The transgenic maize plant cell of embodiment 1a, 1b, 1c, 1d, 1e, 1f,or 1g, wherein said INIR12 transgenic locus comprises the DNA moleculeset forth in SEQ ID NO: 2, 3, 4, 5, 6, 29, 43, 44, 45, or 47 or whereinsaid MIR162 transgenic locus is set forth in SEQ ID NO:1 or SEQ IDNO:46, is present in seed deposited at the ATCC under accession No.PTA-8166, is present in progeny thereof, is present in allelic variantsthereof, or is present in other variants thereof.

9. A transgenic maize plant part comprising the maize plant cell of anyone of embodiments 1a, 1b, 1c, 1d, 1e, 1f, 1g, 2, 3, 4, 5, 6, 7, or 8,wherein said maize plant part is optionally a seed.

10. A transgenic maize plant comprising the maize plant cell of any oneof embodiments 1a, 1b, 1c, 1d, 1e, 1f, 1g, 2, 3, 4, 5, 6, 7, 8, or 8.

11. A method for obtaining a bulked population of inbred seed comprisingselfing the transgenic maize plant of embodiment 10 and harvesting seedcomprising the INIR12 transgenic locus from the selfed maize plant.

12. A method of obtaining hybrid maize seed comprising crossing thetransgenic maize plant of embodiment 10 to a second maize plant which isgenetically distinct from the first maize plant and harvesting seedcomprising the INIR12 transgenic locus from the cross.

13. A DNA molecule comprising SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 25, 37, 39, 40, 41, 42, 43, 44, 45, or 47.

14. A processed transgenic maize plant product comprising the DNAmolecule of embodiment 13.

15. A biological sample containing the DNA molecule of embodiment 13.

16. A nucleic acid molecule adapted for detection of genomic DNAcomprising the DNA molecule of embodiment 13, wherein said nucleic acidmolecule optionally comprises a detectable label.

17. A method of detecting a plant cell comprising the INIR12 transgeniclocus of any one of embodiments 1 a, b, c, d, e, or f to 8, comprisingthe step of detecting DNA molecule comprising SEQ ID NO: 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 25, 37, 39, 40, 41, 42, 43, 44, 45,or 47.

18. A method of excising the INIR12 transgenic locus from the genome ofthe maize plant cell of any one of embodiments 5, 6, 7, or 8, comprisingthe steps of:

(a) contacting the edited transgenic plant genome of the plant cell ofembodiment 5, 6, 7, or 8 with: (i) an RNA dependent DNA endonuclease(RdDe); and (ii) a guide RNA (gRNA) capable of hybridizing to the guideRNA hybridization site of the OgRRS and the CgRRS; wherein the RdDerecognizes a OgRRS/gRNA and a CgRRS/gRNA hybridization complex; and,

(b) selecting a transgenic plant cell, transgenic plant part, ortransgenic plant wherein the INIR12 transgenic locus flanked by theOgRRS and the CgRRS has been excised.

19. The method of embodiment 18, wherein the OgRRS is located in a 3′flanking DNA junction polynucleotide and comprises SEQ ID NO: 26, 27, or28 and wherein the CgRRS comprises an insertion or substitution of SEQID NO: 26, 27, or 28 in a 5′ junction polynucleotide of said INIR12transgenic locus.

20. The method of embodiment 19, wherein the insertion and/orsubstitution is in a 5′ junction polynucleotide of the INIR12 transgeniclocus corresponding to at least one of nucleotides 1079 to 1098 of SEQID NO:1 or SEQ ID NO:46.

21. The method of embodiment 19, wherein the CgRRS comprises the DNAmolecule set forth in SEQ ID NO: 37.

20a. A method of modifying a transgenic maize plant cell comprising:obtaining a MIR162 maize event plant cell, a representative sample ofwhich was deposited at the ATCC under accession No. PTA-8166, comprisinga nucleotide sequence comprising a first ZmUbiInt promoter, a vip3Aa19or vip3Aa20 coding region which is operably linked to said promoter, aCaMV 35S terminator element which is operably linked to said vip3Aa19 orvip3Aa20 coding region, a second ZmUbiInt promoter and an operablylinked phosphomannose isomerase coding region, and a nopaline synthaseterminator element; and modifying said nucleotide sequence to eliminatefunctionality of said phosphomannose isomerase coding region and/or tosubstantially, essentially, or completely remove said phosphomannoseisomerase coding region, and optionally to eliminate functionality of,or substantially, essentially, or completely remove, said secondZmUbiInt promoter.

20b. A method of modifying a transgenic maize plant cell comprising:obtaining a MIR162 maize event plant cell, a representative sample ofwhich was deposited at the ATCC under accession No. PTA-8166, comprisinga nucleotide sequence comprising a first ZmUbiInt promoter, a vip3Aa19or vip3Aa20 coding region which is operably linked to said promoter, aCaMV 35S terminator element which is operably linked to said vip3Aa19 orvip3Aa20 coding region, a second ZmUbiInt promoter and an operablylinked phosphomannose isomerase coding region, and a nopaline synthaseterminator element; and modifying said nucleotide sequence tosubstantially, essentially, or completely remove said phosphomannoseisomerase coding region, and optionally substantially, essentially, orcompletely remove said second ZmUniInt promoter.

20c. A method of making transgenic maize plant cell comprising an INIR12transgenic locus comprising:

(a) contacting the transgenic plant genome of a maize MIR162 plant cellwith: (i) a first set of gene editing molecules comprising a firstsite-specific nuclease which introduces a first double stranded DNAbreak in a 5′ junction polynucleotide of an MIR162 transgenic locus; and(ii) a second set of gene editing molecules comprising a secondsite-specific nuclease which introduces a second double stranded DNAbreak between the CaMV35S terminator element and the ZmUbi promoter ofsaid MIR162 transgenic locus which is operably linked to DNA encoding aphosphomannose isomerase (pmi) and a third site specific nuclease whichintroduces a third double stranded DNA break between the DNA encodingthe pmi and DNA encoding the nopaline synthase (nos) terminator elementof said MIR162 transgenic locus; and

(b) selecting a transgenic maize plant cell, transgenic maize callus,and/or a transgenic maize plant comprising an INIR12 transgenic locuswherein one or more nucleotides of said 5′ junction polynucleotide havebeen deleted and/or substituted, wherein the first ZmUbiInt promoter,the vip3Aa19 or vip3Aa20 coding region which is operably linked to thefirst ZmUbiInt promoter, the CaMV 35S terminator element which isoperably linked to said vip3Aa19 or vip3Aa20 coding region, and the nosterminator element of said MIR162 transgenic locus are present, andwherein DNA of said MIR162 transgenic locus comprising a second ZmUbiIntpromoter and an operably linked phosphomannose isomerase coding regionis absent, thereby making a transgenic maize plant cell comprising anINIR12 transgenic locus.

21. The method of embodiment 20c, comprising:

(a) contacting the transgenic plant genome of a maize MIR162 plant cellwith: (i) a first set of gene editing molecules comprising a firstsite-specific nuclease which introduces a first double stranded DNAbreak between nucleotide residues corresponding to nucleotide number1079 to 1098 of SEQ ID NO:1 or SEQ ID NO:46; and (ii) a second set ofgene editing molecules comprising a second site-specific nuclease whichintroduces a second double stranded DNA break between nucleotideresidues corresponding to nucleotide number 5838 to 5858 of SEQ ID NO:1or SEQ ID NO:46 and a third site specific nuclease which introduces athird double stranded DNA break between nucleotide residuescorresponding to nucleotide number 9040 to 9105 of SEQ ID NO:1 or SEQ IDNO:46; and

(b) selecting a transgenic maize plant cell, transgenic maize plantcallus, and/or a transgenic maize plant wherein one or more nucleotidescorresponding to nucleotide number 1081 to 1104 of SEQ ID NO:1 or SEQ IDNO:46 have been deleted and/or substituted, wherein nucleotidescorresponding to at least nucleotide number 5858 to 9040 of SEQ ID NO:1or SEQ ID NO:46 have been deleted and/or replaced, and whereinnucleotides corresponding to at least nucleotide number 1105 to 5837 ofSEQ ID NO:1 or SEQ ID NO:46 are retained.

22. The method of embodiment 20c or 21, further comprising contactingthe transgenic plant genome of the maize MIR162 plant cell with a donorDNA template comprising a cognate guide RNA recognition site (CgRRS),wherein said CgRRS optionally comprises a polynucleotide set forth inSEQ ID NO: 26, 27, 28, or 37; and selecting a transgenic plant cellwherein said CgRRS has integrated into and/or replaced one or morenucleotides corresponding to at least one of nucleotides 1079 to 1098 ofSEQ ID NO:1 or SEQ ID NO:46.

23. The method of any one of embodiments 20c or 21, wherein the geneediting molecules comprise: (i) a zinc finger nuclease; (ii) a TALEN;and/or (iii) an RNA dependent DNA endonuclease (RdDe) and a guide RNA.

24. The method of embodiment 23, wherein the RNA dependent DNAendonuclease (RdDe) comprises a Cas12a RdDe and wherein the guide RNA ofsaid first set of gene editing molecules comprises SEQ ID NO: 20, theguide RNA of said second set of gene-editing molecules comprises SEQ IDNO: 21, and the guide RNA of said third set of gene-editing moleculescomprises SEQ ID NO: 23.

25. The method of any one of embodiments 20a, b, or c to 24, furthercomprising the step of regenerating transgenic maize plant callus and/ora transgenic maize plant comprising the modification or the INIR12transgenic locus from said transgenic maize plant cell selected in step(c).

26. The method of any one of embodiments 20a, b, or c to 25, furthercomprising the step of harvesting a transgenic maize plant seedcomprising the modification or the INIR12 transgenic locus from thetransgenic maize plant comprising the modification or the INIR12transgenic locus.

27. A transgenic maize plant cell comprising a modification or an INIR12transgenic locus made by the method of any one of embodiments 20a, b, orc to 25.

28. Transgenic maize plant callus comprising a modification or an INIR12transgenic locus made by the method of any one of embodiments 20a, b, orc to 25.

29. A transgenic maize plant comprising a modification or an INIR12transgenic locus made by the method of any one of embodiments 20a, b, orc to 25.

30. A transgenic maize plant seed comprising a modification or an INIR12transgenic locus made by the method of embodiment 26.

EXAMPLES Example 1. Application of a Cas12a and Guide RNAs to Change orExcise the 5′-T-DNA Junction Sequence in the MIR162 Event

The MIR162 5′-junction sequence shown in FIG. 4 is flanked by threeCas12a recognition sequences, gRNA-1 (SEQ ID NO: 20), gRNA-2 (SEQ ID NO:21), and gRNA-3 (SEQ ID NO: 22) that can be used to modify some of the5′ junction sequence or eliminate most of it. There are four possibleiterations of this approach. The first two depend on gRNA-1 and gRNA-3alone to disrupt the MIR162 5′-junction sequence. The second two combinegRNA-2 with either gRNA-1 or gRNA-3 to eliminate most of the MIR162junction sequence. In certain instances, gRNA-1 (SEQ ID NO: 20) is usedto modify the 5′ DNA junction polynucleotide and obtain a modified 5′junction polynucleotide comprising SEQ ID NO: 39 or 40.

The Cas12a nuclease and the single or combined gRNAs are introduced intothe MIR162 event. This can be accomplished in different ways that arefamiliar to those with ordinary skill in the art. The first is to encodeexpression of the Cas12a nuclease and gRNA(s) on a T-DNA and transformit into the MIR162 event via Agrobacterium-mediated transformation.Alternatively, the T-DNA can be transformed into any convenient maizeline, and then crossed with the MIR162 event to combine the Cas12aribonucleoprotein expressing T-DNA with the MIR162 event. The Cas12anuclease and gRNAs can also be assembled in vitro then delivered toMIR162 explants as ribonucleoprotein complexes using a biolisticapproach (Svitashev et al., 2016; doi: 10.1038/ncomms13274). Also, aplasmid encoding a Cas12a nuclease and the gRNA(s) can be delivered toMIR162 explants using a biolistic approach. This will produce plantcells that have a high likelihood of incurring mutations that disruptthe MIR162 junction sequence. To use the Agrobacterium approach a binaryvector that contains a strong constitutive expression cassette like theZmUbi1 promoter::ZmUbi1 terminator driving Cas12a, a PolII or PolIIIgene cassette driving the Cas12a gRNA(s) and a CaMV 35S:PAT:NOS or othersuitable plant selectable marker is constructed. An expression cassettedriving a fluorescent protein like mScarlet may also be useful to theplant transformation process. Constructs are transformed intoAgrobacterium strain LBA4404.

Maize transformations are performed based on published methods (Ishidaet. al, Nature Protocols 2007; 2, 1614-1621). Briefly, immature embryosfrom inbred line GIBE0104, approximately 1.8-2.2 mm in size, areisolated from surface sterilized ears 10-14 days after pollination.Embryos are placed in an Agrobacterium suspension made with infectionmedium at a concentration of OD 600=1.0. Acetosyringone (200 μM) isadded to the infection medium at the time of use. Embryos andAgrobacterium are placed on a rocker shaker at slow speed for 15minutes. Embryos are then poured onto the surface of a plate ofco-culture medium. Excess liquid media is removed by tilting the plateand drawing off all liquid with a pipette. Embryos are flipped asnecessary to maintain a scutelum up orientation. Co-culture plates areplaced in a box with a lid and cultured in the dark at 22° C. for 3days. Embryos are then transferred to resting medium, maintaining thescutellum up orientation. Embryos remain on resting medium for 7 days at27-28° C. Embryos that produce callus are transferred to Selection 1medium with 7.5 mg/L phosphinothricin (PPT) and cultured for anadditional 7 days. Callused embryos are placed on Selection 2 mediumwith 10 mg/L PPT and cultured for 14 days at 27-28° C. Growing calliresistant to the selection agent are transferred to Pre-Regenerationmedia with 10 mg/L PPT to initiate shoot development. Calli remain onPre-Regeneration media for 7 days. Calli beginning to initiate shootsare transferred to Regeneration medium with 7.5 mg/L PPT in Phytatraysand cultured in light at 27-28° C. Shoots that reach the top of thePhytatray with intact roots are transferred to Shoot Elongation mediumprior to transplant into soil and gradual acclimatization to greenhouseconditions.

When a sufficient amount of viable tissue is obtained, it can bescreened for mutations at the MIR162 junction sequence, using aPCR-based approach. One way to screen is to design DNA oligonucleotideprimers that flank and amplify the MIR162 junction plus surroundingsequence. For example, the primers (5′-tttgcatcattggtgtcatcagttttt-3′;SEQ ID NO: 30) and (5′-tttcccgccttcagtttaaactatcag-3′; SEQ ID NO: 31)will produce a ˜310 bp product that can be analyzed for edits at thetarget site. The size of this product will vary based on the nature ofthe edit. Amplicons can be sequenced directly using an ampliconsequencing approach or ligated to a convenient plasmid vector for Sangersequencing. Those plants in which the MIR162 5′-junction sequence isdisrupted are selected and grown to maturity. The DNA encoding theCas12a reagents can be segregated away from the modified junctionsequence in a subsequent generation.

Example 2. Insertion of a CgRRS Element in the 5′-Junction of the MIR162Event

This example describes the construction of plant expression vectors forAgrobacterium mediated maize transformation. Two plant gene expressionvectors are prepared. Plant expression cassettes for expressing aBacteriophage lambda exonuclease, a bacteriophage lambda beta SSAPprotein, and an E. coli SSB are constructed essentially as set forth inUS Patent Application Publication 20200407754, which is incorporatedherein by reference in its entirety. A DNA sequence encoding a tobaccoc2 nuclear localization signal (NLS) is fused in-frame to the DNAsequences encoding the exonuclease, the bacteriophage lambda beta SSAPprotein, and the E. coli SSB to provide a DNA sequence encoding the c2NLS-Exo, c2 NLS lambda beta SSAP, and c2 NLS-SSB fusion proteins thatare set forth in SEQ ID NO: 135, SEQ ID NO: 134, and SEQ ID NO: 133 ofUS Patent Application Publication 20200407754, respectively, andincorporated herein by example. DNA sequences encoding the c2 NLS-Exo,c2 NLS lambda beta SSAP, and c2NLS-SSB fusion proteins are operablylinked to a OsUBI1, ZmUBI1, OsACT promoter and a OsUbi1, ZmUBI1, OsACTpolyadenylation site respectively, to provide the exonuclease, S SAP,and SSB plant expression cassettes.

A donor DNA template sequence (SEQ ID NO: 32) that targets the 5′-T-DNAjunction of the MIR162 event for insertion of a 27 base pairheterologous sequence, that is identical to a Cas12a recognition site atthe 3′-junction of the MIR162 T-DNA insert, by HDR is constructed. Thedonor DNA template sequence includes a replacement template with desiredinsertion region (27 base pair long sequence of SEQ ID NO: 27) flankedon both sides by homology arms about 500-635 bp in length. The homologyarms match (i.e., are homologous to) gDNA (genomic DNA) regions flankingthe target gDNA insertion site. The replacement template regioncomprising the donor DNA template is flanked at each end by DNAsequences identical to the MIR162 5′ polynucleotide sequence recognizedby an RNA-guided nuclease and one or more gRNA(s) (e.g. gRNAs comprisingSEQ ID NO: 20, 21, and 22). In certain cases, a deletion is made in thetargeted MIR162 5′ polynucleotide sequence (e.g., using gRNAs comprisingSEQ ID NO: 20 and 21 in combination or by using gRNAs comprising SEQ IDNO: 21 and 22 in combination).

A plant expression cassette that provides for expression of theRNA-guided sequence-specific Cas12a endonuclease is constructed. A plantexpression cassette that provides for expression of a guide RNAcomplementary to sequences adjacent to the insertion site (e.g. gRNAscomprising SEQ ID NO: 20, 21, and 22) is constructed. An Agrobacteriumsuperbinary plasmid transformation vector containing a cassette thatprovides for the expression of the phosphinothricin N-acetyltransferasesynthase (PAT) protein is constructed. Once the cassettes, donorsequence and Agrobacterium superbinary plasmid transformation vector areconstructed, they were combined to generate two maize transformationplasmids.

A maize transformation plasmid is constructed with the PAT cassette, theRNA-guided sequence-specific endonuclease cassette, the guide RNAcassette, and the MIR162 5′-junction polynucleotide donor DNA templatesequence into the Agrobacterium superbinary plasmid transformationvector (the control vector).

A maize transformation plasmid is constructed with the PAT cassette, theRNA-guided sequence-specific endonuclease cassette, the guide RNAcassette, the SSB cassette, the lambda beta SSAP cassette, the Exocassette, and the MIR162 5′-junction polynucleotide donor DNA templateinto the Agrobacterium superbinary plasmid transformation vector (thelambda red vector).

All constructs are delivered from superbinary vectors in Agrobacteriumstrain LBA4404.

Maize transformations are performed based on published methods (Ishidaet. al, Nature Protocols 2007; 2, 1614-1621). Briefly, immature embryosfrom inbred line GIBE0104, approximately 1.8-2.2 mm in size, areisolated from surface sterilized ears 10-14 days after pollination.Embryos are placed in an Agrobacterium suspension made with infectionmedium at a concentration of OD₆₀₀=1.0. Acetosyringone (200 μM) is addedto the infection medium at the time of use. Embryos and Agrobacteriumare placed on a rocker shaker at slow speed for 15 minutes. Embryos arethen poured onto the surface of a plate of co-culture medium. Excessliquid media is removed by tilting the plate and drawing off all liquidwith a pipette. Embryos are flipped as necessary to maintain a scutelumup orientation. Co-culture plates are placed in a box with a lid andcultured in the dark at 22° C. for 3 days. Embryos are then transferredto resting medium, maintaining the scutellum up orientation. Embryosremain on resting medium for 7 days at 27-28° C. Embryos that producedcallus are transferred to Selection 1 medium with 7.5 mg/Lphosphinothricin (PPT) and cultured for an additional 7 days. Callusedembryos are placed on Selection 2 medium with 10 mg/L PPT and culturedfor 14 days at 27-28° C. Growing calli resistant to the selection agentare transferred to Pre-Regeneration media with 10 mg/L PPT to initiateshoot development. Calli remained on Pre-Regeneration media for 7 days.Calli beginning to initiate shoots are transferred to Regenerationmedium with 7.5 mg/L PPT in Phytatrays and cultured in light at 27-28°C. Shoots that reached the top of the Phytatray with intact roots areisolated into Shoot Elongation medium prior to transplant into soil andgradual acclimatization to greenhouse conditions.

When a sufficient amount of viable tissue is obtained, it can bescreened for insertion at the MIR162 junction sequence, using aPCR-based approach. The PCR primer on the 5′-end can be5′-ttttatgtattatttggtccctaca-3′ (SEQ ID NO: 33) and the PCR primer onthe 3′-end is 5′-gtcgacggcgtttaacaggctggca-3′ (SEQ ID NO: 34). Theseprimers that flank donor DNA homology arms are used to amplify theMIR162 5′-junction sequence. The correct donor sequence insertion willproduce a 1579 bp product. Amplicons can be sequenced directly using anamplicon sequencing approach or ligated to a convenient plasmid vectorfor Sanger sequencing. Those plants in which the MIR162 5′ junctionpolynucleotide sequence now contains the intended CgRRS (e.g., Cas12arecognition sequence in SEQ ID NO: 37) are selected and grown tomaturity. The T-DNA encoding the Cas12a reagents can be segregated awayfrom the modified junction sequence in a subsequent generation. Theresultant INIR12 transgenic locus comprising the CgRRS and OgRRS (e.g.which each comprise SEQ ID NO: 27 and an operably linked PAM site) canbe excised using Cas12a and a suitable gRNA which hybridizes to DNAcomprising SEQ ID NO: 27 at both the OgRRS and the CgRRS. An example ofan INIR12 locus comprising the intended CgRRS in SEQ ID NO: 37 isprovided as SEQ ID NO: 44. Another example of an INIR12 locus comprisingthe intended CgRRS in SEQ ID NO: 37 is provided as SEQ ID NO: 47 and isillustrated in FIG. 6.

Example 3. Deletion of the MIR162 PMI Gene Cassette

The ZmUbi1::PMI coding sequence in MIR162 transgenic maize performs nouseful function with respect to field productivity. It can be removedusing a Cas12a-mediated genomic DNA deletion approach. The procedurecalls for creating an Agrobacterium transformation vector encoding theCas12a nuclease, the MIR162 PMI 5′ guide RNA(5′-taattcctaaaaccaaaatccag-3′; SEQ ID NO: 23), the MIR162 PMI 3′ guideRNA (5′-ttgccaaatgtttgaacgatctg-3′; SEQ ID NO: 24), and a plantselectable marker gene.

A binary vector that contains a strong constitutive expression cassettelike the ZmUbi 1 promoter::ZmUbi1 terminator driving Cas12a, a PolII orPolII gene cassette driving the Cas12a gRNAs and a CaMV 35S:PAT:NOS orother suitable plant selectable marker is constructed. An expressioncassette driving a fluorescent protein like mScarlet may also be usefulto the plant transformation process and included in the binary vector.

The aforementioned binary vector is transformed into maize using theprocedure essentially as outlined in Example 1. The regenerated plantscan be screened with the primer set below to identify individuals thathave lost the ZmUbi1::PMI fragment. The primers span 3820 bases in theintact insert. If both cuts occur and the ends are ligated together,this will produce a ˜555 bp amplicon. This is verified by DNA sequenceanalysis. The primer set includes 162-PMI-ampseq-5′(5′-ggcaacaacctgtacggcggcccga-3′; SEQ ID NO: 35) and 162-PMI-ampseq-3′(5′-gttgccttcagaccatggcggacgt-3′; SEQ ID NO: 36).

Example 4. Introduction of a CgRRS into an INIR12 Maize Plant Comprisinga Deletion of the MIR162 ZmUbi1::PMI Fragment

Maize plants comprising the deletion of the MIR162 ZmUbi1:PMI fragmentare subjected to the procedures for integration of the SEQ ID NO: 32donor DNA template set forth in Example 2 to provide for a resultantINIR12 transgenic locus comprising the CgRRS and OgRRS (e.g. which eachcomprise SEQ ID NO: 27 and an operably linked PAM site) where theZmUbi1::PMI fragment is absent. This resultant INIR12 transgenic locuscan be excised using Cas12a and a suitable gRNA which hybridizes to DNAcomprising SEQ ID NO: 27 at both the OgRRS and the CgRRS.

The breadth and scope of the present disclosure should not be limited byany of the above-described embodiments.

What is claimed is:
 1. A transgenic maize plant cell comprising atransgenic locus set forth in SEQ ID NO:
 47. 2. A transgenic maize plantseed comprising a transgenic locus set forth in SEQ ID NO:
 47. 3. Atransgenic maize plant comprising a transgenic locus set forth in SEQ IDNO:
 47. 4. A method for obtaining a bulked population of seed comprisingselfing the transgenic maize plant of claim 3 and harvesting transgenicseed comprising the transgenic locus set forth in SEQ ID NO: 47.